Agradecemos Sarah Wu e Camille Graham para o desenvolvimento de fases iniciais do ensaio frio sonda, a Bolsa de Centro Bloomington Drosophila para as populações da mosca, e membros do laboratório Galko pela leitura crítica do manuscrito. Este trabalho foi financiado pelo NIH NRSA (NIH F31NS083306) para HNT, e pelo NIH R01NS069828, R21NS087360 e uma Universidade do Texas MD Anderson Clark Fellowship em pesquisa básica para MJG.

1. Preparação de Larvas <ol><li> Levante ações ou cruzamentos genéticos em uma incubadora de 25 ºC. <ol><li> Se o cultivo de uma cruz, use 20-25 fêmeas virgens e 15-20 machos por frasco contendo media milho mosca regular. Permitir fêmeas põem ovos por aproximadamente 48 h antes de transferi-los para um novo frasco de alimentos. </li></ol></li><li> 4-5 dias após a postura de ovos, recolher 3 rd instar larvas do genótipo desejado por esguichando suavemente uma corrente de água para a comida mole e larvas, e despejar o conteúdo em, um prato de Petri limpo de tamanho médio (60 mm x 15 milímetros). </li><li> Em seguida, delicadamente classificar larvas por tamanho usando uma pinça ou um pincel para impedir maior &quot;vagando 3º instar&quot; ou pequenas larvas doentia de ser testado. Rejeitar larvas contendo quaisquer marcadores ou equilibradores genéticos indesejados. </li><li> Tenha um colega de laboratório pratos rótulo ou recipientes onde as larvas classificadas serão colocados de modo que o experimentador pode ser &quot;cego&quot; ao excondição perimental ou genótipo do larvas se for o caso. O experimentador pode aplicar etiquetas descodificados aos dados recolhidos após a experiência utilizando uma chave gerada por um laboratório-membro. </li><li> Usando um scoopula, transferir uma quantidade do tamanho de dime-de alimento fresco para um (35 milímetros x 10 mm ou 60 mm x 15 mm) limpa, marcada placa de Petri cheia de meio caminho com água à temperatura ambiente. </li><li> mover-se suavemente as larvas classificadas na alimentos (para evitar a fome ou dessecação se o teste é antecipado para tomar mais do que 20 min) usando uma pinça ou pincel. </li></ol> 2. Fria Probe Assay <ol><li> Ligar a unidade de sonda fria permitindo um min poucos que arrefeça para a temperatura desejada. Se definir a temperaturas mais baixas, a condensação irá provavelmente formar ao longo da sonda. </li><li> Enxugar qualquer excesso de humidade com um laboratório limpar antes da aplicação para a larva. Quando não está em uso imediato, lugar isolante tampa em ambos sonda para isolar e manter a ponta limpa e evitar danos. </li><li> Coloque um meio 3 rd instar larva sobre uma folha fina de escuro vinil móvel (tipicamente, usar uma pequena peça cortada a partir de uma pasta do caderno) sob um microscópio de campo brilhante. Os auxiliares de vinil preto em contraste visualização e em movimento a larva sem tocá-lo para alinhar adequadamente com a sonda. </li><li> Ajustar o microscópio (ver Tabela de Materiais) e unidade de luz (ver Tabela de Materiais) de brilho médio (50-75% de brilho máximo) para proporcionar o contraste e a impedir que a larva de secar demasiado depressa. </li><li> Descarte qualquer larvas que não primeira exposição locomoção peristáltico normal, como eles poderiam confundir os resultados. A larva deve ser húmido a partir da placa de Petri de água de outro modo a larva vai ficar com o vinil inibir locomoção normal. A água não deve poça em torno da larva. </li><li> Avançar a sonda com a mão em direcção a larva em um ângulo de 90º em relação ao eixo ântero-posterior do corpo, usando o vinilo para mover a larva em CRposição rect (ver Figura 1A). </li><li> Orientar a ponta da sonda a colocar suavemente através da superfície dorsal meados da larva com a sonda a aproximadamente um ângulo de 45º para a platina do microscópio. </li><li> No momento do contacto da sonda, iniciar um temporizador de laboratório. Aplicar pressão descendente suficiente para recuar ligeiramente a superfície da cutícula larvar permitindo ainda para a frente ou para trás, movimento. </li><li> Manter a sonda no lugar até 10 s ou até que uma resposta comportamental evocado a frio é observado - o que ocorrer primeiro. Em seguida, remover a sonda. </li><li> Grave a resposta comportamental observada ea latência de resposta. Larvas que não respondem dentro de 10 s são considerados &quot;não-respondedores&quot;. Latência também podem ser gravadas para respondedores e usadas para medir as alterações em resposta robustez / amplitude. </li><li> Descarte larva e preparar a próxima. </li><li> Repita os passos 2,3-2,11 até que o número desejado de larvas de teste é alcançada (três conjuntos de n = 20-40 larvas foram used aqui). </li></ol>

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Um pedido de patente dos Estados Unidos (CL) está pendente no projeto da sonda fria. Informações adicionais sobre detalhes de números de peça de sonda fria e construção, bem como consultoria adicional no desenho das ferramentas será fornecido mediante solicitação.

O ensaio aqui descrito pode ser utilizado para avaliar quantitativamente e qualitativamente nocicepção ou sensibilização nociceptiva em larvas de várias origens genéticas, as influências ambientais, e / ou condições induzidas por danos. Uma vez que este ensaio permite a aplicação de um estímulo de foco frio, com esta ferramenta pode-se avaliar a função de um subconjunto de neurónios sensoriais periféricos especificamente em resposta a temperaturas frias. Curiosamente, estes comportamentos evocou a frio p...

Drosophila tem provado ser altamente útil para a identificação de novos genes e conservada circuitos neuronais subjacentes a comportamentos complexos. Moscas fornecer um conjunto de ferramentas genéticas sofisticado e um sistema nervoso simplificado que permite a manipulação de precisão genética e neuronal 1, 2, 3, 4 para dissecar as bases celulares e moleculares da nocicepção 5, 6, 7. As larvas são particularmente úteis para estas análises, dado que os ensaios comportamentais para toque suave 8, 9, 10, o calor nocivo 11, 12, 13 e sensação mecânica de estímulos nocivos 4, </sup&gt; 11 já foram estabelecidos, ea cutícula larval transparente permite imagens ao vivo ou fixo da epiderme e neurônios sensoriais subjacentes. Recentemente, um ensaio para frio nocivo também foi desenvolvido 7, que descrevemos em mais detalhes aqui. Usando uma sonda de frio fino, cónico de ponta, mostra-se que as larvas de Drosophila exibem um conjunto de comportamentos reactivos específicos do frio, distinto de comportamentos observados durante a locomoção normais, seguindo toque suave, ou após dura mecânica ou alta estímulos temperatura 7, 8, 11 . Os comportamentos específicos de frio incluem uma contracção robusta de corpo inteiro (CT), um aumento de 45-90º os segmentos posteriores (RP) e um aumento simultâneo do segmentos anterior e posterior em forma de U (US). A prevalência destes comportamentos aumenta com a diminuição da temperatura mas cada picos a slevemente diferentes temperaturas frias. Um trabalho recente sugere que as respostas CT são mediadas por diferentes neurónios sensoriais periféricos do que aqueles que respondem ao calor nocivo ou estímulos mecânicos dura 7. Muito como nociceptores vertebrados, Drosophila dendrítica múltipla (MD) neurónios sensoriais periféricos têm estruturas dendríticas complexos que arborize sobre a epiderme 1. neurónios MD estão presentes em cada segmento do corpo das larvas, que se projecta seus axónios para o cordão nervoso ventral 14. neurónios sensoriais md são separados em quatro classes diferentes (I-IV), com base em morfologia dendrítica e têm diferentes funções sensoriais 4, 9, 10, 15, 16, 17. Embora os neurónios da classe IV, são necessárias para as respostas de rolo lateral do corpo larvaisa temperaturas elevadas ou estímulos mecânicos dura 4, classe III são necessários para os neurónios respostas suave de toque, 9 e 10 não são apenas activados por frio, mas também são necessárias para as respostas comportamentais evocadas frios-7. Ambas as classes III e IV neurónios utilizar canais discretos do receptor transiente potencial (TRP) para facilitar as respostas comportamentais a estímulos 9, 10, 17, 19 nocivo 7, 11, 18 e não nocivo. Além disso, nocicepção larval é sensibilizado lesão seguinte, nos níveis celular 20 e comportamentais 12, 21. O ensaio aqui descrito permite a quantification de qualquer normal, ou potencialmente alterados respostas comportamentais a temperaturas baixas que variam de frio nocivo (≤ 10 ºC), arrefecer inócuo (11-17 ° C), a temperaturas ambiente (18-22 ° C). As temperaturas frias utilizados neste ensaio são capazes de activar directamente classe III neurónios sensoriais, provocando robustos, reprodutíveis e cálcio aumenta as respostas comportamentais evocadas a frio, que pode ser qualitativamente e quantitativamente analisadas 7. Este ensaio pode ser aplicado a virtualmente qualquer larvas de genótipo, bem como para as larvas expostas a diversas condições ambientais (nutrição alterada, ferimentos, agentes farmacológicos) para determinar os factores genéticos e ambientais que têm impacto sobre a nocicepção frio, sensibilização nociceptiva ou plasticidade nociceptiva. Dado que thermosensation é ubíqua em muitas espécies, este ensaio proporciona uma ferramenta valiosa para o estudo da nocicepção e pode descobrir novos alvos gicos ou interacções neuronais que irão melhorarnossa compreensão da nocicepção vertebrados. A sonda fria com especificação (ver sonda fria, Tabela de Materiais) utiliza uma temperatura de circuito fechado controlado dispositivo Peltier, o qual arrefece o eixo de alumínio e ponta cónica através de condução térmica. Um termistor é incorporado no interior da ponta cónica alumínio relata a temperatura em tempo real na unidade de controlo. Um dissipador de calor e do ventilador estão ligados ao módulo termoeléctrico, para regular a carga do efeito Peltier calor (Qc) de modo que o intervalo de temperatura desejado de (22-0 ° C) pode ser obtida (ver a Unidade de Controlo térmica, Tabela de Materiais). O estímulo frio nocivo da ponta da sonda a frio é aplicada à mão à linha média dorsal, ao segmento (s) equidistante das extremidades anterior e posterior (aproximadamente segmento A4, ver Figura 1A) da larva. Em resposta a estímulos frios, geralmente larvas produzir um dos três comportamentos evocado a frio a cerca de 10 s de corte: um corpo completocontracção (CT), um aumento de 45-90º segmentos anterior e posterior em forma de U (US), ou um aumento dos segmentos posteriores (PR) (descritos nos resultados). Nenhum desses comportamentos são realizadas durante a locomoção peristáltico normal ou comportamento de forrageamento. Estes comportamentos são também distintas das respostas de toque suave e a resposta de rolamento aversivo a alta temperatura ou estulos mecicos nocivos.

<table><tbody><tr><td>Cold Probe</td><td>Pro-Dev Engineering</td><td>Custom-built on demand</td><td>Part numbers and construction details can be provided on request</td></tr><tr><td>Thermal Control Unit</td><td>TE Technology</td><td>Custom Built enclosure</td><td>Part numbers and construction details can be provided on request</td></tr><tr><td>Zeiss Stemi 2000 microscope</td><td>Zeiss</td><td>NT55-605</td><td></td></tr><tr><td>Fiber-Lite MI-150 High Intensity Illuminator</td><td>Dolan-Jenner Industries.</td><td>A20500</td><td></td></tr><tr><td>Schott Dual Gooseneck 23 inch Fiber Optic Light Guide</td><td>Schott North America, Inc.</td><td>Schott A08575</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>FS-1670</td><td>Used to sort and handle larvae. Be sure to smooth and blunt forceps tips slightly to lower the risk of accidently puncturing or injuring the larvae</td></tr><tr><td>Paintbrush</td><td>Dick Blick Art Materials</td><td>06762-1002</td><td>Used to sort and handle larvae. It is helpful if the paintbrush is damp during use.</td></tr><tr><td>35 mm x 10 mm Polystyrene Petri dish</td><td>Falcon</td><td>351008</td><td></td></tr><tr><td>60mm x 10 mm Polystyrene Petri dish</td><td>Falcon</td><td>351007</td><td></td></tr><tr><td>Piece of black vinyl (at least 2 inches x 2 inches)</td><td></td><td></td><td>Used to provide contrast and orient larvae to the cold probe</td></tr><tr><td>Fisherbrand Scoopula Spatula</td><td>Fisher Scientific</td><td>14-357Q</td><td>Used to move food</td></tr><tr><td>Kimtech Science Kimwipes</td><td>Fisher Scientific</td><td>06-666A</td><td>Used to dry the larvae and cold probe if there is excess moisture</td></tr></tbody></table>

novel assay for cold nociception in drosophila larvae

Como organismos perceber e responder a temperaturas nocivos ainda é mal compreendido. Além disso, os mecanismos subjacentes a sensibilização das máquinas sensorial, tal como em doentes com neuropatia periférica ou sensibilização induzida pela lesão, não estão bem caracterizados. O modelo de Drosophila geneticamente tratável foi usado para estudar as células e os genes necessários para a detecção de calor nocivo, que produziu vários genes conservados de interesse. Pouco se conhece no entanto sobre as células e receptores importantes para a detecção de frio nocivo. Embora, a Drosophila não sobreviver à exposição prolongada a temperaturas frias (≤10 ºC), e vai evitar fresco, preferindo temperaturas mais quentes em ensaios de preferência comportamentais, como eles detectam e possivelmente evitar estímulos nocivos frio só recentemente foi investigado. Aqui nós descrever e caracterizar o primeiro frio nocivo (≤10 ºC) ensaio comportamentalDrosophila. Usando esta ferramenta e ensaio, mostramos um investigador como avaliar qualitativamente e quantitativamente comportamentos nociceptivos frias. Isto pode ser feito sob condições de cultura normais / saudáveis, ou presumivelmente no contexto de doença, lesão ou sensibilização. Além disso, este ensaio pode ser aplicado a larvas seleccionados para os genótipos desejados, que pode ter impacto thermosensation, dor, ou sensibilização nociceptiva. Dado que a dor é um processo altamente conservado, utilizando este ensaio para um estudo mais aprofundado nocicepção térmica provavelmente vai recolher compreensão importante de processos de dor em outras espécies, incluindo vertebrados.

Drosophila larvas movimento com um movimento peristáltico que inclui pausas ocasionais, vira a cabeça, e mudanças de direção 22. Em resposta ao pedido focal de um estímulo frio nocivo no entanto, as larvas apresentam um conjunto de comportamentos únicos, ao contrário do rolo de lateral aversiva para calor nocivo e estímulos mecânicos. Estes comportamentos são também diferentes de respostas ao toque suave 8, <...

Watch this Scientific Journal Video about Novel Assay for Cold Nociception in Drosophila Larvae at JoVE.com

Novel Assay for Cold Nociception in Drosophila Larvae

Aqui demonstramos um novo ensaio para estudar nocicepção frio em larvas de Drosophila. Este ensaio utiliza uma sonda de Peltier-construído sob encomenda capaz de aplicar um estímulo frio nocivo focal e resulta em comportamento quanto ao frio quantificáveis. Esta técnica permitirá ainda dissecção celular e molecular da nocicepção frio.

Romance Ensaio para Fria nocicepção em<em&gt; Drosophila</em&gt; Larvas

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

novel-assay-for-cold-nociception-in-drosophila-larvae

Research

JoVE Journal

Neuroscience

Novel Assay for Cold Nociception in Drosophila Larvae

7.7K visualizações.  UT MD Anderson Cancer Center.  Aqui demonstramos um novo ensaio para estudar nocicepção frio em larvas de Drosophila. Este ensaio utiliza uma sonda de Peltier-construído sob encomenda capaz de aplicar um estímulo frio nocivo focal e resulta em comportamento quanto ao frio quantificáveis. Esta técnica permitirá ainda dissecção celular e molecular da nocicepção frio. 

Vídeo: Novel Assay for Cold Nociception in Drosophila Larvae

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 locais e globais de Avaliação nocicepção térmica em<em&gt; Drosophila</em&gt; Larvas

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

Neurociência

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.

Visualização de Propriocepção em<em&gt; Drosophila</em&gt; As larvas e 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 ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

Imunologia e Infecção

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.

Aprendizagem associativa apetitiva olfativa em<em&gt; Drosophila</em&gt; As 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.

Luz Ensaio Preferências para estudar inata e circadiano Photobehavior Regulamentado<em&gt; Drosophila</em&gt; As 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.

A Single-fly Ensaio para comportamento de forrageamento em<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 ...

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

Um ensaio e ferramentas aprimoradas para medir o nocicepção mecânico em larvas de Drosophila

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

Comportamento

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.

Um gradiente de temperatura de ensaio para determinar preferências térmicas de larvas de Drosophila

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

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.

This paper presents methods for optogenetic manipulation in Drosophila melanogaster, utilizing CsChrimson and GtACR2 to activate and silence specific neurons. Four experiments are described to utilize optogenetics to explore thermotactic and gustatory behaviors, providing insights into the underlying neural mechanisms governing these processes.

Ensaios Comportamentais para Manipulação Optogenética de Circuitos Neurais em Drosophila melanogaster

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study ...

Local Heat Probe Assay: A Method to Assess Nociception of High Temperatures in Drosophila Larvae

Source: Chattopadhyay, A., et al. <a href="https://www.jove.com/video/3837/" target="_blank">Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae</a>. J. Vis. Exp. (2012).
Drosophila larvae have dedicated sensory neurons&#8211;nociceptors&#8211;whose response to potentially harmful thermal stimuli triggers aversive&#8211;or so-called &#34;nocifensive&#34;&#8211;behaviors in the animal. This video describes a method to assess thermal nociception by recording larval behavior in response to local application of a heat probe onto the cuticle. This method is, therefore, called the local heat probe assay.

Ensaio de sonda de calor local: um método para avaliar a nocicepção de altas temperaturas em larvas de Drosophila

Source: Chattopadhyay, A., et al. Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae. J. Vis. Exp. (2012).
Drosophila larvae ...

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