Nos gustaría dar las gracias a Astha Maltare para la generación de los datos se arrastran larvas. Nos gustaría agradecer al Dr. Nicolás Lanson Jr. para dar sus comentarios sobre el manuscrito. Este trabajo fue apoyado por el Centro Robert Packard para la ELA en la Universidad Johns Hopkins (para UBP) y la Asociación de Esclerosis Lateral Amiotrófica (UBP), y R01MH083689 de los Institutos Nacionales de Salud Mental (CDN).

A. Ensayo de larvas Crawling 1. Las larvas Colección <ol><li> Establecer una botella de 8 onzas de moscas (machos + 10-15 10-15 hembras). </li><li> Que las moscas ponen sus huevos durante 24 horas, luego de botella clara de las moscas. (Transferencia de los adultos en una nueva botella y repita si es necesario). </li><li> Incubar la botella durante 3-4 días, o hasta que las larvas son visibles. </li><li> Agregar 50 a 100 ml de sacarosa al 20% de la botella con las larvas y deje reposar durante 20 minutos. Las larvas flotan en la parte superior. </li><li> Recoger las larvas con una pipeta serológica de 25 ml con la punta cortada, y el lugar en una cesta de malla. </li><li> Lávese las larvas en la cesta de malla dos veces con agua desionizada H 2 O. Las larvas están listas para los experimentos. </li></ol> 2. Para el tratamiento de las larvas con las drogas <ol><li> Utilizar un pincel para transportar el número deseado de las larvas a un vaso de precipitados de 5 ml que contiene una solución de sacarosa al 5% + fármaco. </li><li> Que las larvas se alimentan por lo menos 15 minutos.</li><li> Vierta tratados con el fármaco larvas en una cesta de malla y enjuague. Ahora están listos para usar. </li></ol> 3. Ensayo de locomotora (medida de la distancia total recorrida o las contracciones de la pared corporal) <ol><li> Use un cepillo para el transporte de larvas de individuo a un: <ol><li> 15 cm de placa de Petri que contenía 2% de agarosa (previamente vertió y se deja endurecer) sobre papel milimetrado con un 0,2 cm 2 cuadrícula. <ol><li> Contar el número de líneas de la cuadrícula cruzado en 1 minuto. </li></ol></li><li> Bien de un plato de vidrio que contiene una disección solución de levadura pasta diluida. <ol><li> Cuente las contracciones peristalsis (anterior pleno movimiento posterior contracción = 1) en un minuto mientras se observa bajo un microscopio de disección. </li></ol></li></ol></li><li> Repita hasta que los números deseados de las larvas han sido contados. </li></ol> B. rápido iterativo geotaxis negativos (ARO) del Protocolo Este ensayo fue De originalmentedescrito por Gargano et al 5. <ol><li> Recoger las moscas recién emergidas adultos de sexo masculino menores de CO 2 anestesia ligera y el lugar en un alimento vial estándar que contiene (o comida + drogas). </li><li> Mantener a las moscas a temperatura ambiente (en la parte superior del banco. ~ 22 ° C) durante 2-3 días para permitir la recuperación de CO 2 (y la acumulación de los niveles del fármaco en estado estacionario en su caso). </li><li> Transferir aproximadamente 25 moscas sin anestesiar a viales de poliestireno preparadas. </li><li> Montar viales con moscas en el aparato de anillo (Figura 1). </li><li> Permita que las moscas se adapte al medio ambiente, sin ser molestado, durante 15-20 minutos. </li><li> En este lugar la cámara digital de tiempo ~ 1 m por delante del aparato (en una plataforma, si es necesario para alinear el centro de la lente a media altura de los viales), el enfoque y zoom con la cámara en el aparato, y establecer un temporizador para 3,0 segundos. </li><li> Con cuidado, sujete el aparato de anillo de modo que no con la mano izquierda a la disturb las moscas, y sostienen que el temporizador con la mano derecha. </li><li> Fuertemente toque el aparato hacia abajo sobre la superficie del banco de tres veces, asegurándose de que el grifo es lo suficientemente duro para tumbar todas las moscas a la parte inferior de los viales. </li><li> Simultáneamente con la terminación de la llave en tercer lugar, iniciar el temporizador de cuenta atrás de 3 segundos. </li><li> En tres segundos, tomar una fotografía. </li><li> Restablecer el temporizador de 1 minuto y comenzar. Durante este tiempo, reajustar la cámara y se centran en el aparato, y establecer otro canal del temporizador durante tres segundos. </li><li> Después de 1 minuto, repita los pasos 1.7-1.10 </li><li> Después de un total de 5-6 ensayos, subir imágenes a un ordenador y usar el visor de imágenes preferida para abrir y marcar la altura promedio subió para cada vial. </li><li> Realizar análisis estadístico de los diferentes grupos que comparan la altura media subió. </li></ol> C. El cortejo y apareamiento de ensayo <ol><li> A primera hora de la mañana, claros y productoras de botellas de FLIes para ser utilizado. </li><li> En el transcurso del día (cada 3-4 horas), recoger recién emergidas machos sexualmente ingenuos y mujeres: <ol><li> Coloque los hombres individualmente en frascos o tubos con el medio. </li><li> Coloque 5-6 hembras juntos por vial / tubo. </li><li> Aislar las moscas recolectadas a 25 ° C en 12 h luz / oscuridad durante 5 días. </li><li> Transferencia de una mujer dentro de la cámara de una rueda de apareamiento. </li><li> Transferencia de un macho en la cámara de una rueda de apareamiento. </li><li> Observar par bajo un microscopio de disección de los siguientes comportamientos: <ol><li> Orientación (los varones se orienta hacia la mujer) </li><li> Al tocar (el macho golpea a la hembra) </li><li> Canción del ala (el macho se extiende y hace vibrar un ala) </li><li> Licking (lame los varones los genitales femeninos) </li><li> Curling (el macho se curva su abdomen debajo de sí mismo) </li><li> Intento de cópula (Curling la actividad al intentar montar a la hembra) </li></ol></li></ol></li><li> Observe si hay una0 minutos o hasta que la copulación con éxito, teniendo en cuenta el momento en que cada comportamiento se produce (latencia), el tiempo total involucrado en el comportamiento de cortejo hasta la copulación (para calcular el índice de noviazgo), así como el número de pares que realizar con éxito un determinado comportamiento ( frecuencia). 100% de pares de tipo salvaje generalmente se aparean dentro de los 5 minutos. </li><li> Calcular un índice de cortejo (IC) al dividir el tiempo en el cortejo, dividido por el tiempo total hasta la cópula. Para los pares de tipo salvaje esto por lo general oscila entre 0.6-0.8. </li></ol> D. Representante Resultados Rastreo de ensayo Larva normal de tipo salvaje vagará ~ 3 cm / minuto, y exhibición del ~ 40-50 contracciones de la pared del cuerpo en un minuto. Recientemente hemos desarrollado un modelo de Drosophila FUS / TLS relacionada con la esclerosis lateral amiotrófica que muestra defecto de larvas arrastrándose, esperanza de vida reducida y adultos escalada deterioro 6. Nos centramos en la expresión of de tipo salvaje y formas mutantes de FUS / TLS para las neuronas motoras (OK-371-GAL4 conductor) y realizó un ensayo de rastreo de las larvas. Como se muestra a continuación, las larvas de tipo salvaje arrastran hasta 12 cm, mientras que la expresión de la FUS de tipo salvaje disminución de la capacidad de las larvas arrastrándose de unos 6 cm. Los animales que expresan la mutación R521C ALS causando en FUS / TLS demostrar un deterioro muy grave en su movimiento de rastreo (Figura 1), el rastreo sólo aproximadamente 1 cm por minuto. Geotaxis negativo del ensayo ANILLO Jóvenes de tipo salvaje moscas adultas deben tener una altura media de subida de ~ 4-5 cm en un período de tiempo de 3 segundos (el tiempo se puede ajustar de 3 segundos para dar cabida a diferentes cepas o los niveles de actividad para definir una altura promedio determinado para una determinada cepa / tratamiento). Las moscas que se mantienen en la parte inferior se le asigna un valor de 0. No se recomienda usar más de 25 moscas por vial, ya que entonces se hace difícil determinar la ubicación de cada individuo mosca para medir la altura. Ndesensibilización o se ha observado menos hasta los 6 ensayos consecutivos espaciados 1 minuto, aparte de que hemos empleado. Es crítico no reutilizar los viales de ensayo de poliestireno en este ensayo después de los conjuntos iniciales de datos son recogidos por moscas nuevas colocadas en viales utilizados no subirá a la misma medida en viales frescos. <img alt="Figura 1" src="/files/ftp_upload/3795/3795fig1.jpg" /> Figura 1. De instalación para el ensayo de ANILLO. La cámara digital se coloca aproximadamente 1 m por delante del aparato que contiene las moscas en los viales de poliestireno, el enfoque y zoom con la cámara en el aparato, y establecer un temporizador de 3.0 segundos. <img alt="Figura 2" src="/files/ftp_upload/3795/3795fig2.jpg" /> Datos de la figura 2. Representativos de la prueba de rastreo utilizando larvas moscas ectópica expresar UAS-FUS WT, y UAS-FUS R521C bajo el control de un controlador de la neurona motora (OK371-GAL4). <pclass = &quot;jove_content&quot;&gt;

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No tenemos nada que revelar.

El comportamiento de Drosophila está estrechamente regulada por factores genéticos y ambientales. Nosotros, y otros, han utilizado previamente los ensayos descritos aquí para recoger datos para examinar los genes relacionados a volar a los comportamientos y las enfermedades neurodegenerativas humanas modeladas en Drosophila 5-19. Para la prueba de rastreo, la selección cuidadosa de 3 de las larvas es un paso crítico. Si el tratamiento con un fármaco, se tardará 10-15 minuto...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Sacarosa </td><td> Fisher Scientific </td><td> S5-500 </td></tr><tr><td> Agarosa </td><td> Invitrogen </td><td> 16500-500 </td></tr><tr><td> 6 oz botella de Drosophila </td><td> Genesee Científico </td><td> 32-130 </td></tr><tr><td> Paint Brush (# 1) </td><td> Ted Pella, Inc. </td><td> 11859 </td></tr><tr><td> Fly componentes de los alimentos </td><td></td><td></td></tr><tr><td> Harina de maíz </td><td> Fisher Scientific </td><td> NC9109741 </td></tr><tr><td> Agar </td><td> Genesee Científico </td><td> 66-104 </td></tr><tr><td> Melaza </td><td> Fisher Scientific </td><td> NC9349176 </td></tr><tr><td> El ácido propiónico </td><td> Acros </td><td> 14930-0010 </td></tr><tr><td> Tegosept </td><td> Ápice </td><td> 20-258 </td></tr><tr><td> Etanol </td><td> PescadorCientífico </td><td> BP2818-4 </td></tr><tr><td> Levadura </td><td> Genesee Científico </td><td> 62-107 </td></tr></tbody></table>

methods to assay drosophila behavior

Drosophila melanogaster, la mosca de la fruta, se ha utilizado para estudiar los mecanismos moleculares de una amplia gama de enfermedades humanas tales como cáncer, enfermedades cardiovasculares y diversas enfermedades neurológicas 1. Hemos optimizado los ensayos de comportamiento simples y robustos para determinar la locomoción de larvas, la capacidad de escalada para adultos (ensayo anillo), y comportamientos de cortejo de Drosophila. Estos ensayos de comportamiento son ampliamente aplicables para el estudio del papel de los factores genéticos y ambientales en el comportamiento de la mosca. Capacidad de rastreo de las larvas puede utilizarse de manera fiable para determinar los cambios en las primeras etapas de las habilidades que se arrastran de las larvas de Drosophila y también para examinar el efecto de las drogas o los genes de enfermedades humanas (en moscas transgénicas) en su locomoción. El ensayo de rastreo de las larvas es más aplicable si la expresión o la supresión de un gen que causa la letalidad de la pupa o adulto, ya que estas moscas no sobreviven a la edad adulta que de otro modo podría ser evaluada. Este básico de unssay también puede ser utilizado en conjunción con la luz brillante o estrés adicionales para examinar las respuestas de comportamiento en larvas de Drosophila. El comportamiento de cortejo ha sido ampliamente utilizado para investigar las bases genéticas de la conducta sexual, y también se puede utilizar para examinar la actividad y la coordinación, así como el aprendizaje y la memoria. El comportamiento de cortejo de Drosophila implica el intercambio de diferentes estímulos sensoriales como visuales, auditivas, y las señales de quimiosensoriales entre hombres y mujeres que llevan a una compleja serie de conductas motoras bien caracterizados que culminaron en la cópula exitosa. Los ensayos tradicionales para adultos de escalada (geotaxis negativas) son el trabajo tedioso, intensiva, y requiere mucho tiempo, con una variación significativa entre los diferentes ensayos 2-4. El rápido iterativo geotaxis negativos (RING) ensayo de 5 tiene muchas ventajas sobre los protocolos más ampliamente utilizados, proporcionando un enfoque de rendimiento reproducible, sensible y de alta para cuantificar la locomotora de adultos y negativos b geotaxisehaviors. En el ensayo de ANILLO, varios genotipos o tratamientos de drogas se puede probar de forma simultánea con gran número de animales, con el enfoque de alto rendimiento por lo que es más susceptible para los experimentos de detección.

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Methods to Assay Drosophila Behavior

Drosophila melanogaster es un sistema de genética y de comportamiento modelo manejable que se ha utilizado para entender las bases moleculares y celulares de muchos procesos biológicos importantes para más de un siglo 1. Drosophila ha sido bien aprovechado para hacerse una idea de la base genética del comportamiento de las moscas.

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 ...

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Methods to Assay Drosophila Behavior

42.5K vistas.  Louisiana State University Health Sciences Center. Drosophila melanogaster es un sistema de genética y de comportamiento modelo manejable que se ha utilizado para entender las bases moleculares y celulares de muchos procesos biológicos importantes para más de un siglo 1. Drosophila ha sido bien aprovechado para hacerse una idea de la base genética del comportamiento de las moscas.

Video: Methods to Assay Drosophila Behavior

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Grabaciones solo Sensillum en los insectos Drosophila melanogaster Y Anopheles gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Investigación

Neurociencias

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenético estimulación de comportamiento de escape en<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

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 ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Uso de los canales lineales de agarosa para estudiar<em&gt; Drosophila</em&gt; Comportamiento de las larvas de arrastre

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

Una manera sencilla de medir alteraciones en el comportamiento de búsqueda de recompensa Uso<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Cuantificación de la Drosophila Grooming Comportamiento

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Análisis de alto rendimiento del comportamiento Locomotor en el ensayo de la isla de Drosophila

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

Seguimiento de comportamiento larvario de Drosophila en respuesta a la estimulación de la Optogenetic de las neuronas olfatorias

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Ensayo mecánico de evitación de conflictos para medir el comportamiento del dolor en ratones

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...