Los autores desean agradecer a los Estudios del Desarrollo del Banco hibridoma en la Universidad de Iowa para el envío de anticuerpos. Este trabajo fue apoyado por una beca (N º 29/08) de la Fundación Ciencias de Israel. Como también es apoyado por una beca de investigación de la DFG (Deutsche Forschungsgemeinschaft).

Antes de empezar <ol><li> Crecer las culturas mosca deseados. Conservar los viales con poca gente (alrededor de 30 moscas por vial de 50 ml). Que las moscas ponen sus huevos sólo por un día en cada vial. Esto proporcionará el suministro de larvas de una alimentación suficiente y les permita alcanzar su tamaño máximo antes de arrastrarse fuera de la comida. Conservar los viales en la temperatura adecuada hasta que las larvas comienzan a deambular en la pared del vial. </li><li> Preparar un stock fresca de tampón fosfato salino (PBS) y PBT (0,1% en PBS Tween). Mantenga 10 ml de PBS sobre el hielo. </li><li> Preparar 1-5 ml de fijador (formaldehído al 4% en PBT) y mantener en hielo. Usamos formaldehído embotellada 37-38% como una solución madre. </li></ol> 1. La disección y fijación de las larvas <ol><li> Elige una larva de tercer estadio vagar de la pared del frasco y colocarlo en una caída de 50 l de helado de PBS en una placa de la disección Sylgard (hecho de Sylgard 184 kit de elastómero de silicona, Dow Corning Corporation en unplaca de cultivo de tejidos). </li><li> Mantenga la larva, dorso hacia arriba, cerca de los ganchos de la boca, usando unas pinzas finas (Dumont # 5 fórceps), y se adhieren un alfiler entomológico (minutien, 0,1 mm, acero inoxidable) a través del cerebro de la larva. </li><li> Tome el extremo posterior de la larva con las pinzas, tire suavemente y estirar la larva con suavidad a lo largo. Pegue otro número de identificación de insectos entre los dos espiráculos posteriores. </li><li> Usando tijeras resorte cortar dos incisiones horizontales en la pared del cuerpo (perpendicular al eje antero-posterior), cerca de los pasadores anterior y posterior. </li><li> Usando las tijeras de resorte cortar la pared del cuerpo de larvas a lo largo de la línea media dorsal de la parte anterior a la incisión posterior. </li><li> Retire los órganos internos (intestino, las glándulas salivales, los túbulos de Malpighi, etc) y la tráquea usando unas pinzas finas. Lave suavemente una o dos veces con PBS. </li><li> Agarra las aletas del cuerpo dos de la pared con unas pinzas, se extienden hacia fuera y el pin a la placa con dos pasadores de insectos por cada lado. </li><li> Retire el PBS y añadir 50 l de solución de fijación (4% de formaldehído en PBT). Incubar 20 minutos a temperatura ambiente. </li><li> Retire el fijador. Lavar dos veces con PBS. Tire de los pasadores de los insectos. Con unas tijeras de cortar la cabeza de primavera las larvas y la cola dejando un filete rectangular. Transferir el tejido fijado a un tubo Eppendorf refrigerada con PBS. </li><li> Una vez que un número suficiente de larvas se fijan, uno puede continuar directamente a la tinción de anticuerpos. Si la tinción inmediata no se desea, las larvas fijo se debe lavar 3 veces, 5 minutos cada uno, con 95% de etanol y se almacena en 95% de etanol a -20 ° C. </li></ol> 2. La disección y fijación de las pupas Parte I <ol><li> Asistir a los viales como para la fijación de las larvas, hasta que las larvas comienzan a pupariate. Examine todas las larvas de los viales de horas y la marca que han pupariated. Deje que el pupas marcado el desarrollo de 30-40 horas a 24 ° C, o las horas de 24-27 a 29 ° C. </li><li> Con unas pinzas finas tomar pupas de 20-30 ºe adecuada para su edad y ponerlos en una porcelana oscura de múltiples y plato de disección. Tenga cuidado de no dañar los tejidos de interés. </li><li> Extracto de las pupas de la caja de pupa. Comience por despegar el opérculo y continuará hasta que la pupa es libre. Ponga la pupa en un pozo lleno de PBT. Continúa la extracción de todo el pupas. Pasos 2,4-2,6 debe hacerse en pequeños lotes de cinco pupas a la vez. </li><li> Coloque cinco pupas sobre una superficie plana entre los pozos de la placa de disección. Usando un cuchillo disección micro cortar la punta de la cabeza y el extremo posterior del abdomen (alternativamente, es posible utilizar dos pares de pinzas para desgarrar orificios en ambos extremos de las pupas). </li><li> Mantenga la pupa en el lugar usando unas pinzas finas y usar una jeringa de 1 ml para lavar los órganos internos de la pupa, mediante la inyección de PBT a través de la abertura anterior. </li><li> Lave la disección brevemente pupa por inmersión en un pozo lleno de PBS y avanzar en el fijador frío en un tubo Eppendorf en hielo. </li><li> Incubar toda la noche (ON) a 4 ° C. </li></ol> Parte II <ol start="8"><li> Deseche el fijador y lavar las pupas tres veces, a 5 minutos cada uno, con PBT. Mantenga las pupas lavado en hielo. </li><li> Llene dos pocillos de la placa de disección con PBT. El uso de un polietileno Pasteur pupas pipeta de transferencia varios a uno de los pozos. </li><li> Mover una pupa en un momento en el segundo pozo. El uso de dos pares de pinzas afiladas con puntas perfectamente alineadas, despegue la cutícula transparente pupa del ala: asegurar la pupa en el fondo del pozo con un par de pinzas, y suavemente romper la cutícula con el segundo par de pinzas. Una vez que la cutícula se rompe, quítelo del ala. Tenga cuidado de no desconectar el ala de la pupa. Después de despegar la cutícula de la hoja ala, continuar el pelado de la cutícula de la bisagra ala (donde las alas se encuentran Chos). </li><li> Después de despegar la cutícula del ala, se puede intentar despegar la cutícula pierna de una manera similar.Muchas piernas es probable que se pierde en el proceso, pero a pesar del bajo rendimiento, este paso es muy recomendable, ya que mejora grandemente la tinción de Chos piernas. Los Chos de la haltera y en el abdomen pueden ser visualizados sin más despegando la cutícula. </li><li> Coloque el &#39;pelado&#39; pupa en un tubo Eppendorf lleno de metanol y mantener a temperatura ambiente. Seguir despegando la cutícula de todo el pupas, y añadirlos al mismo tubo. Al menos 10 pupas muy bien pelados se deben recoger para cada tinción. </li><li> Retire el metanol y lavar tres veces, a 5 minutos cada uno, con un 95% de etanol. Pupas fijo se puede mantener durante largos periodos de tiempo en el 95% de etanol a -20 ° C. </li></ol> 3. La inmunotinción de larvas y pupas <ol><li> Lavar el tejido fijado con PBT tres veces, 30 minutos cada una, a temperatura ambiente. Las larvas se pueden lavar con agitación suave en una placa giratoria. Las pupas se lavan sin agitación. </li><li> Vuelva a colocar el PBT con tampón de bloqueo (PBT + 5% nsuero de cabra ormal) y se incuba a 4 ° C. </li><li> Retire el amortiguador de bloqueo y se incuba con anticuerpos primarios (diluida en solución amortiguadora de bloqueo) a 4 ° C. </li><li> Lavar cuatro veces con PBT tal como se describe en el paso 3.1. </li><li> Retire el PBT y se incuba con anticuerpos secundarios (diluida en solución amortiguadora de bloqueo) a 4 ° C. </li><li> Lavar dos veces con PBT y una vez con PBS como se describe en el paso 3,1. </li><li> Vuelva a colocar la PBS por el medio de montaje (Dako Medio fluorescente de montaje (Dako Cytomation, Glostrup, Dinamarca) y se incuba a 4 ° C en ON. </li></ol> 4. Montaje de larvas <ol><li> Las larvas se montan sobre un portaobjetos de microscopio en una gota de medio de montaje con su cutícula hacia abajo y los músculos del cuerpo de la pared hacia arriba. Ponga una pequeña gota de medio de montaje en un cubreobjetos limpio y lo utilizan para cubrir la preparación. No aplique ningún tipo de presión sobre las larvas montado. </li></ol> 5. Montaje de las pupas <ol><li> Prepare dos portaobjetos de microscopiocon una gota de medio de montaje, uno para la disección y el segundo para el montaje. </li><li> Ponga pupas varias en una de las diapositivas. </li><li> Usando tijeras resorte retirar la cabeza y la parte posterior del abdomen. </li><li> Separe la parte dorsal del tórax de la mitad ventral mediante la reducción de entre las alas y las piernas. </li><li> Colocar las partes del cuerpo disecados de la pupa en el portaobjetos de microscopio segundos en una gota de solución de montaje. Asegúrese de que las alas y las piernas se estiran y tratar de minimizar la superposición de los tejidos tanto como sea posible. </li><li> Ponga una gota de medio de montaje en un cubreobjetos y se coloca sobre la muestra. No aplique presión sobre la muestra montada. </li></ol> 6. Los resultados representativos Un ejemplo de inmuno-teñidos de Chos tercera larva se muestra en la Figura 1. Este ejemplo muestra un segmento bien estirado abdominal en la que siete de los ocho Chos son claramente visibles. Las neuronas son de laboratorioELED con MAb22C10 (1:20, obtenido a partir de los Estudios del Desarrollo del Banco hibridoma en la Universidad de Iowa), la tapa, el ligamento, la tapa con el apego y las células del ligamento están marcadas con anti-αTub85E (1:10, Klein et al,. 2010). Secundaria anticuerpos para tinción fluorescente Cy3 fueron, o Cy5-conjugado anti-ratón/conejo (1:200, Jackson ImmunoResearch Laboratories). Las muestras fueron vistos usando microscopía confocal (LSM 510, Zeiss). Un grupo de Chos ala en la vena ventral radial de un 35 horas de edad, crisálidas se muestra en la Figura 2. <img alt="Figura 1" src="/files/ftp_upload/3846/3846fig1.jpg" /> Figura 1 (A) Representación esquemática de los seis tipos de células que constituyen un órgano único: Ch. De fijación de cabeza (CA), la tapa (C), scolopale (S), las neuronas (N), el ligamento (L) y inserción del ligamento (LA ). El órgano LCh5, en el que se agrupan en cinco Chos, se extendía por todo el grupo de músculos laterales transversales (LT1-4). El músculo longitudinal lateral (LL1), ventrales músculos longitudinales (VL1-4) y el músculo lateral oblicua (LO1) también se ilustran. Las células del tendón se muestran como esferas marrones (Tomado de Klein et al., 2010). (B) Un segmento abdominal de un tercio larva vistos en un aumento de 10X. Siete de los ocho Chos presentes en cada segmento abdominal son evidentes: cinco órganos de los laterales que conforman el órgano pentascolopidial (LCh5), un solo órgano lateral (LCh1) y uno de los dos ventrales (Chos VChB). Las neuronas (N) de los Chos se marcan con el marcador neuronal MAb 22C10 (rojo). El ligamento (L), la tapa (C) y las células de fijación (Los Angeles, CA) están marcadas con anti-αTub-85E (azul). Las células de cabeza y de los ligamentos son, además, una etiqueta con un reportero GFP Cho específico de albergar un elemento regulador del locus Dei (Nachman et al, datos no publicados). <img alt="Figura 2" src="/files/ftp_upload/3846/3846fig2.jpg" /> Figura 2. </strong&gt; Los órganos de las alas chordotonal en la vena radial ventral, son vistos con un aumento de 40X. Las neuronas (N) están marcados con los marcadores neuronales 22C10 (Rojo, B). El ligamento (L) y la tapa (C) Las células son co-marcadas con anti-anticuerpos (αTub85E Azul, C) y una Cho-específica del transgén GFP reportero (Nachman et al, datos no publicados).

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El protocolo se describe en este video proporciona una manera de visualizar Chos propioceptivas durante las etapas de larva y pupa. Los estudios sobre la estructura y el desarrollo de Chos propioceptivos han sido hasta ahora limitada principalmente a las fases embrionarias y larvarias discos imaginales. Por lo tanto, muchos aspectos de las etapas posteriores del desarrollo, de Chos tanto larvas y adultos, siguen siendo en gran parte desconocido. El protocolo descrito, junto con herramientas comunes de genética en Drosophila, se puede utilizar para identificar y estudiar nuevos genes y vías que intervienen en las etapas posteriores del desarrollo de Chos. Este protocolo se ha optimizado para la visualización de Cho, pero puede ser adaptado para la tinción de otros tejidos, tales como las alas pupal 6.

<table border="1"><tbody><tr><td> Nombre del reactivo / instrumento </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Dumont # 5 (o # 55) con fórceps, Biologie la punta </td><td> FST </td><td> 11252-20 o 11252-10 (O pinzas similares) </td><td></td></tr><tr><td> Austerlitz pasadores de acero inoxidable de insectos, minutiens 0,1 mm </td><td> Roboz Co Instrumental Quirúrgico </td><td> RS-6083-10 </td><td></td></tr><tr><td> Sylgard 184 kit de silicona de elastómero </td><td> Dow Corning Corporation </td><td> 240.4019862 </td><td></td></tr><tr><td> Vannas micro tijeras (en línea recta, de 7.5 cm, hoja de 3 mm) </td><td> COMO Mdeizintechnik GmbH </td><td> 11-590-00 </td><td> Tijeras Vannas de primavera con especificaciones idénticas se pueden comprar en Roboz instrumental quirúrgico Co. </td></tr><tr><td> Agitador orbital - Rotamax-120 </td><td&gt; Heidolph </td><td> N / A </td><td></td></tr><tr><td> Dako fluorescente Medio de Montaje </td><td> Dako Cytomation, Glostrup, Dinamarca </td><td> DK-5302392 </td><td></td></tr><tr><td> X10 PBS formulación </td><td colspan="2"> 2 gr / KCl iluminado, 2 gr / lit KH 2 PO 4, 80 gr / lit NaCl, 21,7 gr / lit Na 2 HPO4.7H 2 O </td><td> La calidad de PBS es crítico para el éxito de este protocolo </td></tr><tr><td> PBT </td><td colspan="2"> X1 PBS + 0,1% de Tween 20 </td><td></td></tr></tbody></table>

visualization of proprioceptors in drosophila larvae and pupae

La propiocepción es la capacidad de detectar el movimiento o la posición de las partes del cuerpo, respondiendo a los estímulos que surjan dentro del cuerpo. En moscas de la fruta y otros insectos propiocepción es proporcionada por los órganos sensoriales especializados denominados órganos chordotonal (chos) 2. Al igual que muchos otros órganos en la Drosophila, Chos desarrollan dos veces durante el ciclo de vida de la mosca. En primer lugar, las larvas de Chos desarrollan durante la embriogénesis. Entonces, los Chos adultos comienzan a desarrollarse en los discos imaginales de larvas y continuar diferenciándose durante la metamorfosis. El desarrollo de Chos larvas durante la embriogénesis se ha estudiado ampliamente 10,11,13,15,16. La pieza central de cada uno de Cho es una unidad sensorial compuesto de una neurona y una célula scolopale. La unidad sensorial se estira entre dos tipos de células accesorias que se adhieren a la cutícula a través de células especializadas de la epidermis de fijación 1,9,14. Cuando se mueve una larva de la mosca, el desplazamiento relativo de la ecélulas pidermal apego lleva a una extensión de la unidad sensorial y la consiguiente apertura de determinados receptores potenciales transitorios (vanilloides TRPV) canales en el segmento exterior de la dendrita 8,12. La señal provocada se transfiere luego a la locomotora circuito central generador de patrones en el sistema nervioso central. Chos múltiples han sido descritos en la mosca adulta 7. Estos se encuentran cerca de las articulaciones de los apéndices de moscas adultas (patas, alas y cabestros) y en el tórax y el abdomen. Además, varios cientos de Chos en conjunto forman el órgano de Johnston en la antena para adultos que transducen acústica en energía mecánica 3,5,17,4. En contraste con el amplio conocimiento sobre el desarrollo de Chos en etapas embrionarias, se sabe muy poco acerca de la morfología de estos órganos durante las etapas larvales. Además, con la excepción de Chos femorales 18 y el órgano de Johnston, nuestro conociGE sobre el desarrollo y la estructura de Chos en la mosca adulta es muy fragmentaria. A continuación se describe un método para la tinción y visualización de Chos en larvas de tercer estadio y pupas. Este método puede ser aplicado junto con herramientas genéticas para caracterizar mejor la morfología y entender el desarrollo de las diversas Chos en la mosca.

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Visualization of Proprioceptors in Drosophila Larvae and Pupae

Un método para inmunoteñir y visualizar los órganos chordotonal en larvas y pupas de<em&gt; Drosophila melanogaster</em&gt; Se describe.

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

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

visualization-of-proprioceptors-in-drosophila-larvae-and-pupae

Research

JoVE Journal

Neuroscience

Visualization of Proprioceptors in Drosophila Larvae and Pupae

11.9K vistas.  Technion-Israel Institute of Technology. Un método para inmunoteñir y visualizar los órganos chordotonal en larvas y pupas de Drosophila melanogaster Se describe.

Video: Visualization of Proprioceptors in Drosophila Larvae and Pupae

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

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

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

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

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

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

Investigación

Neurociencias

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

Métodos locales y globales de evaluación de nocicepción térmica en<em&gt; Drosophila</em&gt; Las larvas

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

Apetitivo aprendizaje asociativo olfativa en<em&gt; Drosophila</em&gt; Las larvas

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Ensayo Preferencia luz para estudiar innata y circadiano Photobehavior Regulado de<em&gt; Drosophila</em&gt; Las larvas

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Optogenética perturbación de la actividad neuronal con la iluminación láser en semi-intacta<em&gt; Drosophila</em&gt; Las larvas in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

Forrajeo Protocolo Ruta de longitud de<em&gt; Drosophila melanogaster</em&gt; Las larvas

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Ensayo novedoso para la nocicepción en frío<em&gt; Drosophila</em&gt; Las larvas

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Un análisis de gradiente de temperatura para determinar las preferencias térmicas de larvas de Drosophila

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

Optimización del proceso de preparación de muestras para la microscopía electrónica de transmisión de uniones neuromusculares en larvas de Drosophila

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

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.