Damos las gracias a Christian Landry para el calor diseño de la sonda, Daniel Babcock para el desarrollo de la prueba de fuego de la sonda de larvas, Sean Sweeney, por sugerir el ensayo de calor de placas, el Bloomington Drosophila Stock Center para las poblaciones de mosca, y los miembros Galko de laboratorio para la lectura crítica del manuscrito. Este trabajo fue apoyado por el NIH R01 NS069828 de MJG y un NIH MARC U-STAR Training Grant (T34GM079088 a la Universidad de Houston-Downtown Los eruditos de la Academia) para acceso de las minorías a las carreras de investigación (AVG).

<ol>
	<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, 799-806 (2009).</li><li>Tracey, W. D., Wilson, R. I., Laurent, G., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=painless,+a+Drosophila+gene+essential+for+nociception.">painless, a Drosophila gene essential for nociception.</a> Cell. 113, 261-273 (2003).</li><li>Oswald, M., Rymarczyk, B., Chatters, A., Sweeney, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+thermosensitive+escape+behavior+in+Drosophila+larvae.">A novel thermosensitive escape behavior in Drosophila larvae.</a> Fly (Austin). 5, 17810 (2011).</li><li>Grueber, W. B., 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=Tiling+of+the+Drosophila+epidermis+by+multidendritic+sensory+neurons.">Tiling of the Drosophila epidermis by multidendritic sensory neurons.</a> Development. 129, 2867-2878 (2002).</li><li>Moore, A. W., 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=hamlet,+a+binary+genetic+switch+between+single-+and+multiple-+dendrite+neuron+morphology.">hamlet, a binary genetic switch between single- and multiple- dendrite neuron morphology.</a> Science. 297, 1355-1358 (2002).</li><li>Gao, F. B., Brenman, J. E., 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=Genes+regulating+dendritic+outgrowth,+branching,+and+routing+in+Drosophila.">Genes regulating dendritic outgrowth, branching, and routing in Drosophila.</a> Genes Dev. 13, 2549-2561 (1999).</li><li>Walters, E. T., Illich, P. A., Weeks, J. C., Lewin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defensive+responses+of+larval+Manduca+sexta+and+their+sensitization+by+noxious+stimuli+in+the+laboratory+and+field.">Defensive responses of larval Manduca sexta and their sensitization by noxious stimuli in the laboratory and field.</a> J. Exp. Biol. 204, 457-469 (2001).</li><li>Woolf, C. J., Ma, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptors--noxious+stimulus+detectors.">Nociceptors--noxious stimulus detectors.</a> Neuron. 55, 353-364 (2007).</li><li>Hwang, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptive+neurons+protect+Drosophila+larvae+from+parasitoid+wasps.">Nociceptive neurons protect Drosophila larvae from parasitoid wasps.</a> Curr. Biol. 17, 2105-2116 (2007).</li><li>Xiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-avoidance-mediating+photoreceptors+tile+the+Drosophila+larval+body+wall.">Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall.</a> Nature. 468, 921-926 (2010).</li><li>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=Projections+of+Drosophila+multidendritic+neurons+in+the+central+nervous+system:+links+with+peripheral+dendrite+morphology.">Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology.</a> Development. 134, 55-64 (2007).</li><li>Sweeney, N. T., Li, W., Gao, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+single+neurons+in+vivo+reveals+specific+roles+of+flamingo+in+neuronal+morphogenesis.">Genetic manipulation of single neurons in vivo reveals specific roles of flamingo in neuronal morphogenesis.</a> Dev. Biol. 247, 76-88 (2002).</li><li>Kernan, M., Cowan, D., Zuker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+mechanosensory+transduction:+mechanoreception-defective+mutations+of+Drosophila.">Genetic dissection of mechanosensory transduction: mechanoreception-defective mutations of Drosophila.</a> Neuron. 12, 1195-1206 (1994).</li><li>Nitabach, M. N., Blau, J., Holmes, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+silencing+of+Drosophila+pacemaker+neurons+stops+the+free-running+circadian+clock.">Electrical silencing of Drosophila pacemaker neurons stops the free-running circadian clock.</a> Cell. 109, 485-495 (2002).</li></ol>

No tenemos nada que revelar.

Los ensayos descritos aquí se pueden utilizar para evaluar cualitativa y cuantitativamente las larvas de los diferentes genotipos de respuesta a los estímulos nocivos térmicos. Una característica principal de la sonda de ensayo de calor es que el estímulo se da sólo en un solo locus. Se supone que esto da lugar a tiro de sólo un pequeño subconjunto de la clase IV de las neuronas en el segmento de contacto de la sonda y tal vez los de los segmentos adyacentes de inmediato 11. Debido a la estimulación local, el ensayo de la sonda de calor imita la experiencia común sensorial de la detección de un estímulo nocivo que se localiza en un cuerpo en particular la región, como una mano en contacto con una estufa caliente. Una desventaja del ensayo de sonda de calor es que tiene alguna variabilidad de usuario a usuario que probablemente se puede atribuir a tres factores: i) la presión con la que el usuario aplica la sonda a la larva, ii) la ubicación exacta de la sonda en la larva relativa a las neuronas subyacentes nociceptivos, y iii) la precisa angLe en el que la sonda contacto con la superficie de la larva. Hemos informado anteriormente de una estrategia cuantitativa de las larvas de la categorización en los no respondedores, los respondedores lentos, y personal de respuesta rápida en base a su latencia de retirada a una temperatura determinada 1. Aquí nos informe sobre la capacidad de respuesta de larvas a temperaturas aún más altas. Curiosamente, nos encontramos con que hay un techo para las larvas las respuestas nociceptivas térmicas y que este límite máximo se encuentra entre 52 y 54 ° C. Esto puede indicar que las larvas no poseen un potencial de receptor transitorio (TRP) de canal capaz de compuerta a temperaturas superiores a 52 º C. Alternativamente, se podría sugerir que las neuronas o los músculos utilizados para iniciar o llevar a cabo la respuesta del motor se daña antes de que puedan funcionar en la retirada aversivo. También presenta un análisis diferente de la amplitud de la retirada de respuesta usando ya sea el número de rollos como un indicador de la &quot;robustez&quot; de la respuesta. Ingenuamente, unoCabría esperar que estos parámetros se incrementaría con el aumento de temperatura o tiempo de estimulación. Sorprendentemente, se encuentra que este no es el caso. Las larvas se estimularon durante un tiempo más largo a una temperatura en el extremo inferior del rango nocivo (42 ° C) muestran más rollos y más tiempo dedicado rodadura que las larvas probaron a altas temperaturas (48-52 ° C). Esto sugiere que, dentro de la ventana de temperatura nociva es principalmente la duración de la exposición que determina la amplitud de la respuesta. Dado que las larvas se exponen a temperaturas altamente nocivas (48-52 ° C) responden en promedio muy rápidamente, no exhiben como muchos rollos como larvas expuestas a una temperatura de menos nocivo para una longitud de tiempo más largo. El análisis de la amplitud de la respuesta que aquí añade una nueva dimensión cuantitativa a lo largo de la cual los diferentes genotipos o manipulaciones ambientales se pueden comparar. Una característica principal de la placa de ensayo de calor es que implica una exposición global a la nocivacalor. Como tal, es más parecido al animal sentado en un caldero de calefacción que tocar una estufa caliente. Aunque no está claro cuando una larva puede experimentar un estímulo nocivo a nivel mundial en la naturaleza, en el laboratorio de las respuestas de comportamiento a esta exposición mundial son más complejas que las observadas después de la estimulación local. Una fuerza de la placa de ensayo de calor, también señalado por otros 3, es que tiene poco de usuario a usuario variabilidad desde tocar la larva no es un componente del protocolo. La única variación importante parece estar en la definición de cuándo comienza cada comportamiento, y esto puede ser minimizado con visión repetida / familiaridad. Una diferencia interesante entre los ensayos son las temperaturas a las que las conductas aversivas comenzar. Estos son mucho más baja en el ensayo de calor de placas que con la sonda de calor. El comportamiento exhibido por las larvas preliminar en contacto con la sonda de calor (cabeza y cola de subida) puede ser un correlato de la cabeza del movimiento de piernas observa en ~ 27 ° C en la plataforma de calore ensayo. Es posible que esta respuesta refleja el &quot;malestar&quot; más que &quot;el dolor&quot;. No hemos observado una correlación de los azotes, la confiscación, y la parálisis, incluso a alta (hasta 48 ° C) las temperaturas en el ensayo de la sonda de calor y puede ser que una masa crítica de las neuronas sensoriales disparando desde más de una región del cuerpo es necesaria para que en esos comportamientos. Interesantemente, los comportamientos convulsivos y parálisis se observan a temperaturas (~ 34 a 37 ° C) por debajo del extremo inferior del umbral nociceptivo observado con la sonda de calor que indica que la estimulación global puede implicar suma de las respuestas neuronales que son insuficientes para provocar un comportamiento con locales aplicación de la sonda de calor. Que estas temperaturas son en realidad percibida como nociva para las larvas se apoya en la observación de que las larvas que comienzan el comportamiento parálisis y posteriormente se dejaron recuperar en alimentos mosca no en la mayoría de los casos sobrevivir (Figura 5C). Más apoyo a la tesis de que el calor de la plensayo comió está leyendo las respuestas nociceptivas es el hecho de que el bloqueo de la transmisión sináptica en las neuronas sensoriales nociceptivas conocidos aumenta la latencia de la mayoría de los comportamientos observados (Figura 6). La observación de que no hay aumento en la latencia para el comportamiento de mayor temperatura para batir sugiere que otros neuronas sensoriales que no expresan MD-Gal4 puede ser requerido para este comportamiento. En suma, los dos ensayos implican la exposición de una larva de individuo a un estímulo nocivo térmica de la temperatura se define - la punta caliente de una pequeña sonda de metal en el ensayo local y la inmersión en una gota de agua rápidamente el calentamiento en el ensayo mundial. Las respuestas de comportamiento de las larvas de Drosophila de diferentes orígenes genéticos y / o expuestos a diferentes condiciones ambientales (por ejemplo, más o menos daño a los tejidos), se puede estudiar y cuantificar el uso de estos ensayos. En última instancia, los resultados de estos ensayos nos ayudará a comprender mejor las redes genéticas que controlan nociception en Drosophila y otras especies relacionadas.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Sonda térmica </td><td> Pro-Dev Ingeniería </td><td> Custom-construido sobre la demanda </td><td> La información de contacto puede ser proporcionada en la solicitud de </td></tr><tr><td> Baño seco Incubadora </td><td> Fisher Scientific </td><td> 11-718 </td><td> Un bloque de calentamiento sólido y un bloque de calentamiento con los pozos de 16 mm </td></tr><tr><td> Leica DFC290 12v/400mA Cámara en color </td><td> Leica Microsystems </td><td> 12730080 </td><td> Cualquier equivalente cámara hará. </td></tr><tr><td> Leica MZ6 microscopio </td><td> Leica Microsystems </td><td> El número de parte para el cuerpo de zoom MZ6 (portadora óptica) es 10445614 </td><td></td></tr><tr><td> Schott Ace Modulamp Unidad </td><td> Schott North America, Inc. </td><td> A20500 </td><td></td></tr><tr><td> Schott doble cuello de cisne de 23 pulgadas de fibra óptica de guía de luz </td><td> Schott North America, Inc. </td><td> Schott A08575 </td><td></td></tr><tr><td> Unidad de control térmico </td><td> TSCI corp. </td><td> Custom Built </td><td> Los detalles pueden ser disponibles bajo petición </td></tr><tr><td> Zeiss Stemi 2000 microscopio </td><td> Zeiss </td><td> NT55-605 </td><td> Cualquier equivalente microscopiova a hacer. </td></tr><tr><td> Fórceps </td><td> FST </td><td> FS-1670 </td><td></td></tr><tr><td> Malla de 1 mm </td><td> Genesee Científico </td><td> 57-101 </td><td></td></tr><tr><td> Pincel </td><td> Dick Blick Art Materials </td><td> 06762-1002 </td><td></td></tr><tr><td> Reticulante UV </td><td> Fisher Scientific </td><td> 1199289 </td><td></td></tr><tr><td> Recipientes </td><td> Fisher Scientific </td><td> 08-816 </td><td></td></tr><tr><td> Vaso de precipitados de 10 ml </td><td> Fisher Scientific </td><td> 02-540C </td><td></td></tr><tr><td> Éter dietílico </td><td> Fisher Scientific </td><td> E138-500 </td><td></td></tr><tr><td> 35 x 10 mm de poliestireno Petri </td><td> Halcón </td><td> 351008 </td><td> No hemos probado platos alternativos. </td></tr><tr><td> Portaobjetos del microscopio </td><td> Corning </td><td> 26003 </td><td></td></tr><tr><td> Termopar </td><td> Omega Engineering, Inc. </td><td> HH802U </td><td></td></tr><tr><td> Pedazo de vinilo </td><td> Office Depot </td><td> 480009 </td><td></td></tr><tr><td> Microcentrífuga tubo </td><td> Denville CientíficoInc. </td><td> C-2170 </td><td></td></tr></tbody></table>

local and global methods of assessing thermal nociception in drosophila larvae

En este artículo, nos demuestran los ensayos para estudiar la nocicepción térmica en larvas de Drosophila. Un ensayo consiste en la estimulación espacialmente restringido (local) de 1,2 nociceptores térmicos, mientras que la segunda implica una mayor (mundial) la activación de la mayoría o todas las neuronas tipo 3. En conjunto, estas técnicas permiten la visualización y cuantificación de las funciones de comportamiento de Drosophila neuronas sensoriales nociceptivas. La larva de Drosophila es un modelo de sistema establecido para estudiar la nocicepción térmica, una respuesta sensorial a temperaturas potencialmente dañinos que es evolutivamente conservados a través de 1,2 especies. Las ventajas de Drosophila para estos estudios son la relativa simplicidad de su sistema nervioso y la sofisticación de las técnicas genéticas que se pueden utilizar para diseccionar las bases moleculares de la biología subyacente 6.4 En Drosophila, como en todos los metazoos, los response para estímulos nocivos térmicos en general, implica un &quot;nocifensive&quot; retirada de aversión al estímulo presentado 7. Tales estímulos son detectados a través de las terminaciones nerviosas libres o nociceptores y la amplitud de la respuesta organísmica depende del número de nociceptores que reciben el estímulo nocivo 8. En Drosophila, es la clase IV de las neuronas sensoriales arborización dendríticas que detectan los estímulos nocivos térmicos y mecánicos 9, además de su papel recientemente descubierto como fotorreceptores 10. Estas neuronas, las cuales han sido muy bien estudiados en el nivel de desarrollo, se ramifican en la hoja de la barrera epidérmica y hacer contactos con casi todas las células de la epidermis 11,12. El solo axón de cada neurona proyectos de clase IV en el cordón nervioso ventral del sistema nervioso central 11 donde pueden conectarse a las neuronas de segundo orden que se proyectan hacia el cerebro. En condiciones basales, neuro sensorial nociceptivons no se disparará hasta un umbral relativamente alto que se alcanza. Los ensayos descritos aquí permiten que el investigador cuantificar las respuestas de comportamiento o de referencia, presumiblemente, la sensibilización que sobreviene el daño tisular siguiente. Cada ensayo provoca distintas pero relacionadas las respuestas de comportamiento locomotor de estímulos nocivos térmicos y permite al investigador para visualizar y cuantificar distintos aspectos de la nocicepción térmica en larvas de Drosophila. Los ensayos se pueden aplicar a las larvas de los genotipos deseados o de larvas criadas en diferentes condiciones ambientales que puedan afectar la nocicepción. Dado que la nocicepción térmica se conserva en todas las especies, los resultados recogidos de la disección genética en Drosophila es probable que nuestra comprensión de la nocicepción térmica en otras especies, incluyendo los vertebrados.

Watch this Scientific Journal Video about Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae at JoVE.com

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae | Neuroscience | Video

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

En este artículo, nos demuestran los ensayos para estudiar la nocicepción térmica en<em&gt; Drosophila</em&gt; Larvas. Un ensayo consiste en la estimulación espacialmente restringido (local) de los nociceptores térmicos<sup&gt; 1,2</sup&gt; Mientras que el segundo implica una mayor (mundial) la activación de la mayoría o todas estas neuronas<sup&gt; 3</sup&gt;. En conjunto, estas técnicas permiten la visualización y cuantificación de las funciones de comportamiento de<em&gt; Drosophila</em&gt; Las neuronas sensoriales nociceptivas.

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 ...

local-global-methods-assessing-thermal-nociception-drosophila

Research

Education

JoVE Journal

JoVE Core

Biology

Neuroscience

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

Human Biology

Sensory Systems

SCIENTISTS IN ACTION

15.7K vistas.  The University of Texas MD Anderson Cancer Center. En este artículo, nos demuestran los ensayos para estudiar la nocicepción térmica en Drosophila Larvas. Un ensayo consiste en la estimulación espacialmente restringido (local) de los nociceptores térmicos 1,2 Mientras que el segundo implica una mayor (mundial) la activación de la mayoría o todas estas neuronas 3. En conjunto, estas técnicas permiten la visualización y cuantificación de las funciones de comportamie...

Video: Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Las mediciones electrofisiológicas y Análisis de la nocicepción en los bebés humanos

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Investigación

Neurociencias

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Métodos de ensayo a Drosophila Comportamiento

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

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

Visualization of Proprioceptors in Drosophila Larvae and Pupae

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

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

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

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 ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

Métodos electrofisiológicos para medir los niveles de fotopigmento en fotorreceptores de Drosophila

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...