C.M. es apoyado por fondos de la NEI (EY008117, EY010852), NIDCD (DC007864, DC016278) y el NIAID (1DP1AI124453).

<ol>
	<li>Fowler, M. A., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+TRP+channels+and+animal+behavior.">Drosophila TRP channels and animal behavior.</a> Life Sci. 92, 394-403 (2013).</li><li>Palkar, R., Lippoldt, E. K., McKemy, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+cellular+basis+of+thermosensation+in+mammals.">The molecular and cellular basis of thermosensation in mammals.</a> Curr Opin Neurobiol. 34, 14-19 (2015).</li><li>Vriens, J., Nilius, B., Voets, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+thermosensation+in+mammals.">Peripheral thermosensation in mammals.</a> Nat Rev Neurosci. 15 (9), 573-589 (2014).</li><li>Rosenzweig, M., 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=The+Drosophila+ortholog+of+vertebrate+TRPA1+regulates+thermotaxis.">The Drosophila ortholog of vertebrate TRPA1 regulates thermotaxis.</a> Genes Dev. 19, 419-424 (2005).</li><li>Kwon, Y., Shim, H. S., Wang, X., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+thermotactic+behavior+via+coupling+of+a+TRP+channel+to+a+phospholipase+C+signaling+cascade.">Control of thermotactic behavior via coupling of a TRP channel to a phospholipase C signaling cascade.</a> Nat Neurosci. 11, 871-873 (2008).</li><li>Kwon, Y., Shen, W. L., Shim, H. S., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+thermotactic+discrimination+between+the+optimal+and+slightly+cooler+temperatures+via+a+TRPV+channel+in+chordotonal+neurons.">Fine thermotactic discrimination between the optimal and slightly cooler temperatures via a TRPV channel in chordotonal neurons.</a> J Neurosci. 30 (31), 10465-10471 (2010).</li><li>Shen, W. 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=Function+of+rhodopsin+in+temperature+discrimination+in+Drosophila.">Function of rhodopsin in temperature discrimination in Drosophila.</a> Science. 331 (6022), 1333-1336 (2011).</li><li>Sokabe, T., Chen, H. S., Luo, J., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+switch+in+thermal+preference+in+Drosophila+larvae+depends+on+multiple+rhodopsins.">A switch in thermal preference in Drosophila larvae depends on multiple rhodopsins.</a> Cell Rep. 17, 336-344 (2016).</li><li>Ni, 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=The+Ionotropic+Receptors+IR21a+and+IR25a+mediate+cool+sensing+in+Drosophila.">The Ionotropic Receptors IR21a and IR25a mediate cool sensing in Drosophila.</a> Elife. 5, 13254 (2016).</li><li>Luo, 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=Navigational+decision+making+in+Drosophila+thermotaxis.">Navigational decision making in Drosophila thermotaxis.</a> J Neurosci. 30 (12), 4261-4272 (2010).</li><li>Ashburner, M., Golic, K. G., Hawley, R. 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> Drosophila: a laboratory handbook. , (2005).</li></ol>

Los autores no tienen nada que revelar.

Para asegurar el éxito de este protocolo, es importante que tome medidas para obtener el número suficiente de larvas para realizar los experimentos. Estos incluyen la alimentación de las moscas en viales que contienen pasta de levadura para 2-3 d mejorar la puesta de huevos. Los viales necesitan ser colocados en una bandeja que contiene frascos de agua y dentro de una bolsa de plástico transparente, que mantiene la humedad de los alimentos y promueve la efectiva de alimentación por las larvas permitiendo la exposici...

Thermotaxis es empleado por los animales móviles para seleccionar un entorno con las condiciones más favorables1,2,3. Si el clima es excesivamente frío o caliente, este comportamiento es vital para la supervivencia. Además, muchos animales son sensibles a diferencias muy pequeñas de temperatura en la gama cómoda y buscan un entorno con una temperatura ideal. Esto es de particular importancia para los organismos poiquilotérmicas como moscas de la fruta, que equilibre su temperatura corporal con el medio ambiente. Ensayos para controlar larvas thermotaxis han sido instrumentales en identificar y clarificar las funciones de sensores moleculares tales como Drosophila potencial Receptor transitorio (TRP) canales4,5,6, rodopsinas7,8e ionotrópicos del receptor receptores (IRs)9, que dotan a estos animales con sensibilidad de temperatura sobre rangos de temperaturas diferentes.
Una prueba de selección bidireccional proporciona un método para estudiar las preferencias térmicas en larvas6,7. El ensayo consiste en establecer dos zonas de distinta temperatura y permite a los animales seleccionar un lado sobre el otro. Los resultados de pruebas de dos vías de elección pueden ser sólidos, especialmente si son grandes las diferencias de temperatura entre las dos opciones. Además, puesto que cada ensayo implica tabular sólo dos grupos, los datos pueden expresarse como un índice simple preferencia. La facilidad y sencillez de los análisis de dos vías de elección son también susceptibles a las pantallas de la genéticas. Sin embargo, una limitación importante es que muchos experimentos se necesitan para establecer la temperatura preferida de los animales de tipo silvestre o mutantes.
Un análisis de gradiente ofrece la oportunidad de establecer la temperatura preferida en un solo análisis8. Además, a diferencia de la prueba de selección bidireccional, permite la evaluación de la distribución de un grupo de animales, cuando se enfrentan a un rango continuo de temperaturas. Un análisis de gradiente utiliza una placa de Petri y solos animales y es adecuado para caracterizar el comportamiento detallado de animales individuales10. Sin embargo, desde platos de Petri son redondos, los tamaños de las zonas de temperatura varían y son progresivamente más pequeños dependiendo de la distancia desde el centro. Por lo tanto, esta configuración no es ideal para el monitoreo de las selecciones de temperatura de las poblaciones de animales.
Un aparato de gradiente térmico continuo que es adecuado para evaluar las preferencias de la temperatura de los grupos de larvas emplea una arena rectangular y se describe aquí. El aparato es fácil de construir y montar. Además, el gradiente es lineal y es flexible, ya que puede ser utilizado evaluar thermotaxis sobre grandes temperatura oscila entre 10 ° C a 42 ° C. El ensayo es rápido y sencillo de realizar y da datos reproducibles. Además de reportar la temperatura favorecida de larvas, revela las preferencias de la población de animales sobre toda una gama linear en un solo experimento. Debido a estas ventajas, es una excelente opción para la identificación de genes necesarios para thermotaxis.

<table>
	<tbody>
		<tr>
			<td>Gradient assay apparatus</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PolyScience 9106, Refrigerated/Heated 6L Circulating Bath</td>
			<td>Thomas Scientific</td>
			<td>9106</td>
			<td>This model is discontinued. Updated replacement models include: 1186R00 and 1197U04 for 120 V, 60 Hz, or 1184L08 and 1197U04 for 240 V, 50 Hz.</td>
		</tr>
		<tr>
			<td>Aluminum assay plate (for single directional gradient)</td>
			<td></td>
			<td></td>
			<td>Outer size: 14 x 10.1 x 0.9 cm, inner size: 12.9 x 8.7 x 0.8 cm, black anodized.</td>
		</tr>
		<tr>
			<td>Aluminum plate (for bidirectional gradient)</td>
			<td></td>
			<td></td>
			<td>25 x 22 x 0.2 cm, black anodized.</td>
		</tr>
		<tr>
			<td>Aluminum block</td>
			<td></td>
			<td></td>
			<td>Outer size: 25.5 x 5 x 1.4 cm, parameters of inner channels are shown in Figure 1D.</td>
		</tr>
		<tr>
			<td>Connector for aluminum blocks and tubing</td>
			<td>McMaster-Carr</td>
			<td>91355K82</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon Sanitary Silicone Tubing</td>
			<td>Tygon</td>
			<td>57296</td>
			<td>1/4&#34; ID x 3/8&#34; OD x 1/16&#34; wall</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Items and reagents for assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle</td>
			<td>USA Scientific</td>
			<td>17361</td>
			<td>Pestle for 1.5 mL microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Fluke</td>
			<td>51II</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocouple</td>
			<td>Fluke</td>
			<td>K type</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal microplate lid</td>
			<td>Corning</td>
			<td>6980A77</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm dish</td>
			<td>Corning</td>
			<td>9380D40</td>
			<td></td>
		</tr>
		<tr>
			<td>Labeling tape (for bidirectional gradient)</td>
			<td>Fisher Scientific</td>
			<td>15-951</td>
			<td>Fisherbrand labeling tape 2 in x 14 yds</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Invitrogen</td>
			<td>16500500</td>
			<td>Prepare 1% solution</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389-5KG</td>
			<td>Prepare 18% solution right before starting assay</td>
		</tr>
		<tr>
			<td>Paint brush</td>
			<td>Fisher Scientific</td>
			<td>11860</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Denville</td>
			<td>C1062-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Scoopula</td>
			<td>Fisher Scientific</td>
			<td>14-357Q</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL round wide-mouth bottle</td>
			<td>Pyrex</td>
			<td>1395-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (300 mm pore)</td>
			<td>PluriSelect</td>
			<td>43-50300</td>
			<td>Optional item for larvae washing</td>
		</tr>
		<tr>
			<td>Cardboard box (vial tray)</td>
			<td>Genesee Scientific</td>
			<td>FS32-124</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Drosophila food</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td>22,400 mL</td>
		</tr>
		<tr>
			<td>Cornmeal, yellow (extra fine mesh,flocked) 20 kg</td>
			<td>LabScientific Inc.</td>
			<td>NC0535320</td>
			<td>1,609 g</td>
		</tr>
		<tr>
			<td>Brewers yeast 100 lbs</td>
			<td>MP Biomedicals</td>
			<td>ICN90331280</td>
			<td>379 g</td>
		</tr>
		<tr>
			<td>NutriSoy&#174; Soy Flour (10 kg/unit)</td>
			<td>Genesee Scientific</td>
			<td>62-115</td>
			<td>221 g</td>
		</tr>
		<tr>
			<td>Drosophila Agar, Type II (5 kg)</td>
			<td>Genesee Scientific</td>
			<td>66-103</td>
			<td>190 g</td>
		</tr>
		<tr>
			<td>Karo light corn syrup</td>
			<td>Karo</td>
			<td></td>
			<td>1,700 mL</td>
		</tr>
		<tr>
			<td>Methyl 4-hydroxybenzoate (suspend in 200 proof ethanol)</td>
			<td>Sigma Aldrich</td>
			<td>H5501-5KG</td>
			<td>72 g/240 mL</td>
		</tr>
		<tr>
			<td>Propionic acid puriss. p.a.,&#62;99.5% (GC)</td>
			<td>Sigma Aldrich</td>
			<td>81910-1 L</td>
			<td>108 mL</td>
		</tr>
		<tr>
			<td>Phosphoric acid&#160;ACS reagent, &#8805;85 wt. % in H2O</td>
			<td>Sigma Aldrich</td>
			<td>438081-500 mL</td>
			<td>8.5 mL</td>
		</tr>
	</tbody>
</table>

a temperature gradient assay to determine thermal preferences of drosophila larvae

Muchos animales, incluyendo la mosca de la fruta, Drosophila melanogaster, son capaces de discriminantes diferencias minutos en temperatura ambiente, que les permite buscar su paisaje termal preferido. Para definir las preferencias de la temperatura de las larvas sobre un rango lineal definido, hemos desarrollado un ensayo utilizando un gradiente de temperatura. Para establecer un gradiente solo bidireccional, dos bloques de aluminio están conectados con baños independientes, cada una de ellas controla la temperatura de bloques individuales. Los dos bloques establece los límites inferiores y superiores del gradiente. El gradiente de temperatura se establece mediante la colocación de una placa de aluminio recubierto de agarosa sobre los dos bloques de agua controlada para que la placa extiende la distancia entre ellos. Los extremos de la placa de aluminio que se encuentra en la parte superior de los bloques de agua define las temperaturas mínimas y máximas, y las regiones entre los dos bloques forman un gradiente lineal de la temperatura. El análisis de gradiente puede ser aplicado a las larvas de diferentes edades y puede ser usado para identificar a mutantes que exhiben fenotipos, como aquellas con mutaciones que afectan a genes que codifican los canales TRP y opsins, que se requieren para la discriminación de temperatura.

Para establecer un solo-direccionales de 18 ° C - 28 ° C gradiente, fijamos las temperaturas de los dos baños de agua a 16,8 ° C y 31 ° C. Obtenemos las temperaturas a 13 puntos por la medición de la temperatura en 26 posiciones dentro de las porciones superiores e inferiores de todas las 6 zonas, las líneas de frontera entre las zonas y en los extremos de la superficie del gel de agarosa (figura 2, 2E). La distribución de temperatura...

Watch this Scientific Journal Video about A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae at JoVE.com

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Aquí, presentamos un protocolo para determinar la temperatura ambiente preferida de larvas de Drosophila usando un gradiente térmico continuo.

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 ...

a-temperature-gradient-assay-to-determine-thermal-preferences

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

7.5K vistas.  University of California, Santa Barbara. Aquí, presentamos un protocolo para determinar la temperatura ambiente preferida de larvas de Drosophila usando un gradiente térmico continuo.

Video: A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Nanoinjection parejas y Ensayo de electrofisiología para la detección de bioactividad de compuestos utilizando el Drosophila melanogaster Sistema de fibra gigante

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

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 ...

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 ...

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 ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

Un ensayo de una sola mosca por el comportamiento de forrajeo en<em&gt; Drosophila</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

El uso de una Multiplexación Ensayo de la caspasa para determinar la apoptosis en un modelo de célula hipotalámica

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Ensayo de Alto Rendimiento para el Examen de Preferencias de la puesta de huevos individual<em&gt; Drosophila melanogaster</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

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 ...

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

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