El diseño original del compensador de torque se origina con Karl Goetz. La configuración particular en esta presentación es en gran medida, en calidad de préstamo y fue desarrollado originalmente por Martin Heisenberg y el lobo Reinhard. Estoy especialmente en deuda con estas dos personas por su apoyo continuo estímulo y experiencia.

Volar medio La composición de los alimentos mosca es crítico para el aprendizaje (Guo et al, 1996.) <ul><li> 1000 ml de agua </li><li> 180 g de harina de maíz </li><li> De soja 10 g </li><li> 18,5 g de levadura </li><li> 7,5 g de agar </li><li> Melaza 40 g </li><li> Jarabe (remolacha azucarera) 40 g </li><li> Nipagin 2,5 g </li></ul> Cada vial se suministra con un poco de pasta fresca, levadura de vida y un pedazo de papel de filtro para proporcionar una superficie adicional para las moscas y las pupas. Reproducción de moscas y puesta en escena El siguiente procedimiento se realiza todos los días, lo que precisamente realizaron animales criados en la densidad adecuada. Todas las moscas recién ecclosed ya que el procedimiento por última vez el día anterior se recogen para la cría y experimentos. Los más antiguos viales sin ningún tipo de pupas viven restantes se descartan. Cuatro moscas días de edad se suman a un vial de nuevo para la deposición de huevos durante la noche. La densidad de moscas hembra debe ser de aproximadamente 20 por cada vial, ajustado por el tamaño del frasco y se utiliza la cepa. La densidad ideal es aquel que es lo suficientemente alta como para el medio de volar a licuarse durante las etapas de larva y lo suficientemente bajo de tal manera que todas las larvas han empupado antes de la ecclose primeras moscas. Las moscas ponen huevos desde el día anterior se extrae y se desecha. Volar preparación Las moscas se mantienen en el nivel de harina de maíz / melaza medio como se describió anteriormente a 25 ° C y 60% de humedad con un régimen de 12h light/12hr oscuro. Después de una breve inmovilizar 24-48h moscas de edad por el frío de la anestesia, las moscas se pegan (SuperGlue UV de vidrio adhesivo, 505127A, Pacer Technology, Cucamonga, CA., EE.UU.) con la cabeza y el tórax con un gancho de cobre en forma de triángulo (diámetro 0,05 mm ) el día antes del experimento. Los animales son entonces mantenerse aisladas durante la noche en pequeñas cámaras húmedas que contienen unos pocos granos de sacarosa. Aparato El dispositivo central de la puesta en marcha es el compensador de torque (par metro) (Götz, 1964). Que mide el momento angular de una mosca alrededor de su eje vertical del cuerpo, causada por las maniobras de vuelo previsto. La mosca, pegado al anzuelo como se describe anteriormente, se une a la par de metros a través de una pinza para llevar a cabo vuelo estacionario en el centro de un panorama cilíndrico (arena, diámetro 58 mm), que es homogéneamente iluminados por detrás. La fuente de luz es un 100W, 12V de tungsteno-yodo bombilla. Para la iluminación verde y azul de la arena, la luz pasa a través de filtros de banda ancha monocromática Kodak Wratten de gelatina (# 47 y # 99, respectivamente). Los filtros pueden ser intercambiados por un solenoide rápida en 0,1 segundos. Por otra parte, el escenario se ilumina con &quot;luz&quot; al pasar por un filtro azul-verde (Rosco &quot;surfblue&quot; N º 5433), o sin filtro en absoluto. El espectro de transmisión de la Rosco azul-verde filtro utilizado en este estudio es equivalente a la de un filtro BG18 (Schott, Mainz) y constituye un intermedio entre el azul de Kodak y los filtros verdes (Brembs y Hempel de Ibarra, 2006; Liu et al ., 1999). La arena se puede girar alrededor de la mosca con un motor eléctrico controlado por ordenador. En un &quot;simulador de vuelo&quot; la situación, la velocidad angular de la arena es proporcional, pero en contra del par de guiñada de la mosca (acoplamiento factor K =- 11 ° / s • 10-10 Nm). Esto permite a la marcha para estabilizar el panorama y para controlar su orientación angular. Esta virtual &quot;la dirección de vuelo&quot; (es decir, la posición de arena) se graba continuamente a través de un potenciómetro circular (Novotechnik, A4102a306). Un sistema analógico al digital tarjeta convertidora (PCL812; Advantech Co.) se alimenta de la posición de la arena y la señal del par de guiñada en un ordenador que almacena las huellas (frecuencia de muestreo 20 Hz) para su posterior análisis. El castigo se logra mediante la aplicación de calor de un láser ajustable de infrarrojos (825 nm, 150 mW), dirigida por detrás y por encima en la cabeza de la mosca y el tórax. El rayo láser es pulsado (aproximadamente de ancho de pulso de 200ms en ~ 4 Hz) y su intensidad reducida para asegurar la supervivencia de la mosca. Experimentos Patrón de aprendizaje Para los patrones tradicionales de aprendizaje experimento (Dill y Heisenberg, 1995; eneldo et al, 1993, 1995;. Liu et al, 2006;. Liu et al, 1998;. Liu et al, 1999;. Wolf y Heisenberg, 1991) , cuatro negro, en forma de T patrones de orientación alterna (es decir, dos verticales y dos invertida) se espacian uniformemente en la pared de arena (patrón de ancho de ψ = 40 °, la altura de θ = 40 °, ancho de las barras = 14 °, como se ha visto desde la posición de la mosca). Un programa de computadora divide el 360 ° de la arena en 4 cuadrantes · virtual 90, los centros de los cuales se denotan por los patrones. El control de las moscas de la posición angular de los patrones con su par de guiñada (situación simulador de vuelo). Durante el entrenamiento, el castigo calor es contigua a la aparición de una de las orientaciones patrón en el campo visual frontal. Refuerzo de cada patrónsiempre se igualan dentro de los grupos. Durante la prueba, el calor está permanentemente apagado y registró la preferencia de la mosca del patrón. Color de aprendizaje El aprendizaje del color se realiza como se describió anteriormente (Brembs y Heisenberg, 2000; Brembs y Hempel de Ibarra, 2006; Brembs y Wiener, 2006; Wolf y Heisenberg, 1997). El escenario se divide en cuatro cuadrantes · virtual 90, los centros de los cuales se denotan por cuatro franjas verticales idénticos (ancho ψ = 14 °, la altura de θ = 40 °). La mosca es el control de la posición angular de las cuatro barras idénticas a su par de guiñada como se describe para los patrones en forma de T por encima. El color de la iluminación de toda la arena se cambia cada vez que una de las fronteras virtuales cuadrante pasa por un punto frente a la marcha. Durante el entrenamiento, el castigo de calor está supeditada a uno de los dos colores. Durante la prueba, el calor está permanentemente apagado y registró la preferencia de la mosca de color. Por supuesto, los colores se pueden combinar con los patrones para el acondicionamiento del compuesto (Brembs y Heisenberg, 2001). Guiñada aprendizaje par Aprendizaje de desvío de par se realiza como se describió previamente (Brembs y Heisenberg, 2000, Heisenberg y Wolf, 1993). La mosca de la gama espontánea par de guiñada se divide en una &quot;izquierda&quot; y &quot;derecho&quot; de dominio, que corresponde aproximadamente a cualquiera de giros a la izquierda oa la derecha. No hay patrones en la pared de arena. Durante el entrenamiento, el calor se aplica cuando el par de la mosca de guiñada se encuentra en un dominio y se apaga cuando el par pasa a la otra. En las fases de prueba, el calor está permanentemente apagado y la elección de la mosca de la guiñada dominios registrados es par. Aprendizaje compuesto Aprendizaje compuesto es una extensión del aprendizaje par de orientación, como se describió anteriormente (Brembs y Heisenberg, 2000). Básicamente, el aprendizaje y el aprendizaje de orientación par de colores se combinan en un experimento con operante equivalente (par de guiñada) y clásica (colores) predictores. Durante el entrenamiento, la mosca se calienta cada vez que el par de la mosca de guiñada pasa al dominio asociado con el castigo. Cada vez que cambia la marcha dominios desvío de par, no sólo la temperatura sino también la coloración escenario ha cambiado (de verde a azul o viceversa). Por lo tanto, de dominio y el color de orientación par servir como predictores equivalente de calor. En las fases de prueba, el calor está permanentemente apagado y la única opción de la mosca de colores par de guiñada dominios / es registrada. Discusión Esta instalación experimental combina un excelente control sobre las circunstancias experimentales con un organismo modelo genético avanzado. Utilizando los procedimientos descritos en esta presentación, los fundamentos moleculares y neurobiológicos de una variedad de rasgos de comportamiento pueden ser investigados, incluyendo pero no limitado a, los mecanismos de generación de un comportamiento espontáneo, el condicionamiento operante y clásico, el reconocimiento de patrones, la visión del color o el control de curso .

<ol>
	<li>Guo, A., 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=Conditioned+visual+flight+orientation+in+Drosophila;+Dependence+on+age,+practice+and+diet.">Conditioned visual flight orientation in Drosophila; Dependence on age, practice and diet.</a> Learn. Mem.. 3, 49-59 (1996).</li><li>Goetz, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optomotorische+Untersuchung+des+visuellen+Systems+einiger+Augenmutanten+der+Fruchtfliege+Drosophila.">Optomotorische Untersuchung des visuellen Systems einiger Augenmutanten der Fruchtfliege Drosophila.</a> Kybernetik. 2, 77-92 (1964).</li><li>Liu, L., Wolf, R., Ernst, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+generalization+in+Drosophila+visual+learning+requires+the+mushroom+bodies.">Context generalization in Drosophila visual learning requires the mushroom bodies.</a> Nature. 400, 753-756 (1999).</li><li>Brembs, B., Hempel de Ibarra, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+parameters+support+generalization+and+discrimination+learning+in+Drosophila+at+the+flight+simulator.">Different parameters support generalization and discrimination learning in Drosophila at the flight simulator.</a> Learn. Mem. 13, 629-637 (2006).</li><li>Liu, G., 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=Distinct+memory+traces+for+two+visual+features+in+the+Drosophila+brain.">Distinct memory traces for two visual features in the Drosophila brain.</a> Nature. 439, 551-556 (2006).</li><li>Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+organization+of+operant+behavior+as+revealed+in+Drosophila+flight+orientation.">Basic organization of operant behavior as revealed in Drosophila flight orientation.</a> J. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 169, 699-705 (1991).</li><li>Liu, L., 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=Conditioned+visual+flight+orientation+in+Drosophila+melanogaster+abolished+by+benzaldehyde.">Conditioned visual flight orientation in Drosophila melanogaster abolished by benzaldehyde.</a> Pharmacol Biochem Behav. 61, 349-355 (1998).</li><li>Dill, M., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+pattern+memory+without+shape+recognition.">Visual pattern memory without shape recognition.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 349, 143-152 (1995).</li><li>Dill, M., Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+pattern+recognition+in+Drosophila+involves+retinotopic+matching.">Visual pattern recognition in Drosophila involves retinotopic matching.</a> Nature. 365, 751-753 (1993).</li><li>Dill, M., Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+analysis+of+Drosophila+landmark+learning+in+the+flight+simulator.">Behavioral analysis of Drosophila landmark learning in the flight simulator.</a> Learn. Mem. 2, 152-160 (1995).</li><li>Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Space+from+Visual+Motion:+Turn+Integration+in+Tethered+Flying+Drosophila.">Visual Space from Visual Motion: Turn Integration in Tethered Flying Drosophila.</a> Learn. Mem. 4, 318-327 (1997).</li><li>Brembs, B., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+operant+and+the+classical+in+conditioned+orientation+in+Drosophila+melanogaster+at+the+flight+simulator.">The operant and the classical in conditioned orientation in Drosophila melanogaster at the flight simulator.</a> Learn. Mem. 7, 104-115 (2000).</li><li>Brembs, B., Wiener, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+generalization+and+occasion+setting+in+Drosophila+visual+learning.">Context generalization and occasion setting in Drosophila visual learning.</a> Learn. Mem. 13, 618-628 (2006).</li><li>Brembs, B., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioning+with+compound+stimuli+in+Drosophila+melanogaster+in+the+flight+simulator.">Conditioning with compound stimuli in Drosophila melanogaster in the flight simulator.</a> J Exp Biol. 204, 2849-2859 (2001).</li><li>Heisenberg, M., Wolf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sensory-motor+link+in+motion-dependent+flight+control+of+flies.">The sensory-motor link in motion-dependent flight control of flies.</a> Rev. Oculomot. Res. 5, 265-283 (1993).</li></ol>

The authors have nothing to disclose.

Esta instalación experimental combina un excelente control sobre las circunstancias experimentales con un organismo modelo genético avanzado. Utilizando los procedimientos descritos en esta presentación, los fundamentos moleculares y neurobiológicos de una variedad de rasgos de comportamiento pueden ser investigados, incluyendo pero no limitado a, los mecanismos de generación de un comportamiento espontáneo, el condicionamiento operante y clásico, el reconocimiento de patrones, la visión del color o el control de curso .

operant learning of drosophila at the torque meter

Para los experimentos en el medidor de torque, las moscas se mantienen en un medio de vuelo estándar a 25 ° C y 60% de humedad con un régimen de 12h light/12hr oscuro. Un régimen de cría estandarizada asegura la densidad adecuada de larvas y las cohortes de edades similares. Frío anestesiados moscas se pegan con la cabeza y el tórax con un gancho en forma de triángulo el día antes del experimento. Se adjunta a la par de metros a través de una pinza, la mosca intención maniobras de vuelo se mide como el momento angular alrededor de su eje vertical del cuerpo. La mosca se coloca en el centro de un panorama cilíndrico para realizar vuelo estacionario. Un análogo de la tarjeta convertidora de señal digital se alimenta la señal del par de guiñada en un ordenador que almacena la traza para su posterior análisis. El equipo también controla una variedad de estímulos que pueden estar bajo control de la mosca de cerrar el bucle de retroalimentación entre estos estímulos y el seguimiento del par de derrape. El castigo se logra mediante la aplicación de calor de un láser infrarrojo ajustable.

Watch this Scientific Journal Video about Operant Learning of Drosophila at the Torque Meter at JoVE.com

Operant Learning of Drosophila at the Torque Meter

Medir el par de atados de guiñada Drosophila con el medidor de par motor permite que el control neurocientífico exquisito de la naturaleza del estímulo de los animales de experimentación. Junto con las herramientas de genética única disponible en la mosca de la fruta, este paradigma se utiliza para una amplia variedad de la investigación neurobiológica.

El aprendizaje operante de Drosophila en el medidor de torque

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized ...

operant-learning-of-drosophila-at-the-torque-meter

Research

JoVE Journal

Biology

13.4K vistas.  Free University of Berlin. Medir el par de atados de guiñada Drosophila con el medidor de par motor permite que el control neurocientífico exquisito de la naturaleza del estímulo de los animales de experimentación. Junto con las herramientas de genética única disponible en la mosca de la fruta, este paradigma se utiliza para una amplia variedad de la investigación neurobiológica.

Video: Operant Learning of Drosophila at the Torque Meter

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Cargando Drosophila terminales nerviosas con indicadores de calcio

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Investigación

Biología

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2 estimulación mediada de los potenciales sinápticos en Drosophila Las uniones neuromusculares

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Marcaje fluorescente de Drosophila Estructuras del Corazón

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Imágenes in vivo de larvas de Drosophila intactos a nivel sub-celular Resolución

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Comparación cuantitativa de Cis-Elemento regulador (CRE) Las actividades de transgénicos Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombinería homólogos Construcciones recombinación en<em&gt; Drosophila</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

El cultivo de células humanas epiteliales nasales en la interfase aire líquido

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Disección y montaje de<em&gt; Drosophila</em&gt; Pupal discos Eye

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Estudiar mitocondrial Estructura y función en<em&gt; Drosophila</em&gt; Los ovarios

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...