Os autores gostariam de agradecer ao Banco do Desenvolvimento de Estudos Hybridoma da Universidade de Iowa para enviar-nos anticorpos. Este trabalho foi suportado por uma concessão (n º 29/08) de The Science Foundation Israel. AS também é apoiado por uma bolsa de pesquisa da DFG (Deutsche Forschungsgemeinschaft).

Antes de começar <ol><li> Crescer as culturas mosca desejados. Manter os frascos uncrowded (aproximadamente 30 moscas por 50 frasco ml). Deixe as moscas depositam ovos para apenas um dia em cada frasco. Isto irá fornecer as larvas abastecimento alimentar suficiente e permitir-lhes atingir o tamanho máximo antes rastejando para fora do alimento. Mantenha os frascos dentro da temperatura adequada até larvas de terceiro estádio começam a vagar na parede do frasco. </li><li> Preparar um estoque fresca de solução salina tamponada com fosfato (PBS) e PBT (0,1% de Tween em PBS). Manter 10 ml de PBS em gelo. </li><li> Preparar 1-5 ml de fixador fresco (formaldeído a 4% em PBT) e mantê-lo em gelo. Usamos formaldeído 37-38% engarrafada como uma solução estoque. </li></ol> 1. Dissecção e fixação de larvas de terceiro estádio <ol><li> Escolha um errante larva de terceiro instar a partir da parede do frasco e colocá-la numa queda de 50 ul de PBS gelado sobre uma placa de dissecação Sylgard (feita de elastómero 184 Sylgard kit de silicone, Dow Corning Corporation, em umatecido prato de cultura). </li><li> Mantenha a larva lado, dorsal para cima, perto dos ganchos da boca, utilizando fórceps finos (Dumont # 5 fórceps), e ater um pino de insectos (minutien, 0,1 mm, de aço inoxidável), através do cérebro a larva da. </li><li> Agarre a extremidade posterior da larva com a pinça, puxe-a delicadamente e esticar a larva suavemente longitudinalmente. Fure outro pino inseto entre os dois espiráculos posteriores. </li><li> Tesoura de mola usando cortar duas incisões horizontais na parede do corpo (perpendicular ao eixo anterior-posterior), perto de os pinos anterior e posterior. </li><li> Usando as tesouras de mola cortar a parede do corpo larval ao longo da linha média dorsal a partir do anterior à incisão posterior. </li><li> Remover órgãos internos (intestino, glândulas salivares, túbulos de Malpighi, etc) ea traquéia usando uma pinça fina. Lavar suavemente uma vez ou duas vezes com PBS. </li><li> Pegue os dois retalhos de parede do corpo com pinças, esticar-los e fixá-los à placa através de dois pinos de insetos para cada lado. </li><li> Remover o PBS e adicionar 50 uL de solução de fixação (formaldeído a 4% em PBT). Incubar 20 minutos à temperatura ambiente. </li><li> Remover o fixador. Lavar duas vezes com PBS. Retire os alfinetes de insetos. Usando uma tesoura de mola cortou a cabeça ea cauda larval deixando um filete retangular. Transferir o tecido fixado a um tubo de Eppendorf, refrigerada com PBS. </li><li> Quando um número suficiente de larvas são fixos, pode-se continuar directamente para a coloração de anticorpos. Se a coloração imediata não é desejado, as larvas fixa deverá ser lavado 3 vezes, 5 minutos cada, com etanol a 95% e armazenadas em etanol a 95% a -20 ° C. </li></ol> 2. Dissecção e Fixação de pupas Parte I <ol><li> Atender aos frascos como para a fixação das larvas, até larvas de terceiro estádio começam a pupariate. Examinar os tubos todos os larvas hora e marca que pupariated. Permitir que a pupas marcada para desenvolver 30-40 horas a 24 ° C, ou 24-27 horas a 29 ° C. </li><li> Utilizando uma pinça fina escolher 20-30 pupas de thidade e adequado e coloque-os num prato de porcelana escura multi-bem de dissecação. Tenha cuidado para não danificar os tecidos de interesse. </li><li> Extraia o pupas do caso pupal. Comece descascando o opérculo e continue até a pupa é gratuito. Coloque a pupa em um poço cheio de PBT. Continue a extrair todos pupas. Passos 2,4-2,6 deve ser feito em pequenos lotes de cinco pupas de cada vez. </li><li> Coloque cinco pupas sobre uma superfície plana entre os poços do prato de dissecação. Usando uma faca dissecção micro corte da ponta da cabeça e da ponta posterior do abdómen (alternativamente, é possível usar dois pares de pinças para rasgar furos em ambas as extremidades de pupas). </li><li> Mantenha a pupa no local usando fórceps finos e utilizar uma seringa de 1 mL para lavar os órgãos internos do pupa, por injecção de PBT através da abertura anterior. </li><li> Lava-se a dissecados brevemente pupa por imersão em um poço cheio com PBS e movê-lo para o fixador frio em um tubo de Eppendorf mantido em gelo. </li><li> Incubar durante uma noite (ON) a 4 ° C. </li></ol> Parte II <ol start="8"><li> Descarte o fixador e lavar as pupas três vezes, 5 minutos cada, com PBT. Mantenha a pupas lavado em gelo. </li><li> Encher dois poços do prato de dissecação com PBT. Usando um polietileno Pasteur pupas transferência pipeta vários a um dos poços. </li><li> Mover uma pupa de cada vez para o segundo poço. O uso de dois pares de pinças com pontas afiadas perfeitamente alinhadas, retire a cutícula transparente pupa da asa: assegurar a pupa para o fundo do poço, utilizando um par de pinças, e suavemente rasgar a cutícula usando o segundo par de pinças. Uma vez que a cutícula estiver rasgado, retire-a da asa. Tenha cuidado para não desconectar a asa da pupa. Depois de descascar a cutícula da lâmina asa, continue descascando a cutícula da dobradiça asa (onde os Chos asas estão localizados). </li><li> Depois de descascar a cutícula asa, uma pode tentar descascando a cutícula perna de uma maneira semelhante.Muitas pernas são susceptíveis de serem perdidos no processo, mas, apesar do baixo rendimento, esta etapa é altamente recomendável uma vez que melhora a coloração de Chos perna. Os Chos do haltere e abdômen pode ser visualizado sem mais descascando a cutícula. </li><li> Coloque a &#39;desenrolada&#39; pupa em um tubo de Eppendorf preenchido com metanol e manter à temperatura ambiente. Continuar descascando a cutícula de toda a pupas, e adicioná-los ao mesmo tubo. Pelo menos 10 pupas bem descascadas devem ser coletadas para cada coloração. </li><li> Remover o metanol e lava-se três vezes, 5 minutos cada, com etanol a 95%. Pupas fixo pode ser mantida por longos períodos de tempo em etanol a 95% a -20 ° C. </li></ol> 3. A imunocoloração de larvas e pupas <ol><li> Lavar o tecido fixado com PBT três vezes, 30 minutos cada, à temperatura ambiente. As larvas podem ser lavados com agitação suave sobre uma placa rotativa. As pupas são lavados sem agitação. </li><li> Substituir o PBT com tampão de bloqueio (PBT + 5% de nsoro de cabra ormal) e incubar ON a 4 ° C. </li><li> Remover o tampão de bloqueio e incubar com anticorpos primários (diluído em tampão de bloqueio) ON a 4 ° C. </li><li> Lavar quatro vezes com PBT como descrito no passo 3.1. </li><li> Remover o PBT e incubar com anticorpos secundários (diluído em tampão de bloqueio) ON a 4 ° C. </li><li> Lavar duas vezes com PBT e uma vez com PBS como descrito no passo 3.1. </li><li> Substituir o PBS com meio de montagem (Dako Médio fluorescente de montagem (Dako Cytomation, Glostrup, Dinamarca) e incubar a 4 ° C em. </li></ol> 4. Montagem de larvas <ol><li> As larvas são montados sobre uma lâmina de microscópio em uma gota de meios de montagem com a sua cutícula virada para baixo e musculatura da parede do corpo voltada para cima. Coloque uma pequena gota de meio de montagem em uma lamínula limpa e usá-lo para cobrir a preparação. Não aplique pressão sobre as larvas montado. </li></ol> 5. Montagem de pupas <ol><li> Preparar duas lâminas de microscópiocom uma gota de meio de montagem, uma para a dissecção ea segunda para a montagem. </li><li> Coloque pupas vários sobre uma das lâminas. </li><li> Usando uma tesoura de mola remover a cabeça ea parte posterior do abdômen. </li><li> Separa-se a meia dorsal do tórax a partir da metade ventral cortando entre as asas e as pernas. </li><li> Colocar as partes do corpo dissecados do pupa sobre a segunda lâmina de microscópio em uma gota de solução de montagem. Certifique-se de que as asas e as pernas estão esticadas para fora e tentar minimizar sobreposição de tecidos, tanto quanto possível. </li><li> Colocar uma gota de meio de montagem numa lamela e colocá-la sobre a amostra. Não aplique pressão sobre a amostra montada. </li></ol> 6. Os resultados representativos Um exemplo de imuno-coradas CHOs de larva de terceiro instar é mostrado na Figura 1. Este exemplo mostra um segmento muito bem esticada abdominal em que sete dos oito CHOs são claramente visíveis. Os neurônios são laboratóriomodelada com MAb22C10 (1:20, obtido a partir do Banco Developmental Hybridoma Estudos da Universidade de Iowa); a tampa, ligamento, tampão de fixação e as células de fixação do ligamento são rotulados com anti-αTub85E (1:10, Klein et ai,. 2010). Os anticorpos secundários para coloração fluorescente foram Cy3, ou Cy5-conjugado anti-mouse/rabbit (1:200, Jackson Immunoresearch Laboratories). As amostras foram visualizadas por microscopia confocal (LSM 510, Zeiss). Um aglomerado de CHOs asa na veia ventral radial de um 35 hr de idade pupa é mostrado na Figura 2. <img alt="A Figura 1" src="/files/ftp_upload/3846/3846fig1.jpg" /> A Figura 1 (A) ilustração esquemática dos seis tipos de células que constituem um órgão Ch único:. De fixação tampa (CA), tampão (C), scolopale (S), neurónio (N), o ligamento (L) e do ligamento de fixação (LA ). O órgão de LCh5, em que cinco CHOs estão agrupados, é esticado em todo o grupo de músculos laterais transversais (LT1-4). O músculo lateral longitudinal (LL1), ventral músculos longitudinais (VL1-4) e do músculo oblíquo lateral (LO1) também são ilustradas. Células do tendão são ilustrados como esferas marrons (tirado de Klein et al., 2010). (B) Um segmento abdominal de uma larva de terceiro instar visto em uma ampliação de 10x. Sete dos oito CHOs presentes em cada segmento abdominal são evidentes: cinco órgãos laterais que juntos formam o órgão pentascolopidial (LCh5), um único órgão lateral (LCh1) e um de dois CHOs ventral (VChB). Os neurónios (N) dos CHOs são rotulados com o marcador neuronal MAb 22C10 (vermelho). O ligamento (L), tampão (C) e as células de fixação (LA, CA) são marcadas com anti-αTub-85E (azul). As células do tampão e do ligamento são adicionalmente marcado com um repórter da GFP específica CHO-abrigando um elemento regulador do locus dei (Nachman et ai, dados não publicados). <img alt="A Figura 2" src="/files/ftp_upload/3846/3846fig2.jpg" /> Figura 2. </strong&gt; Os órgãos asa chordotonal na veia ventral radial são vistos com uma ampliação de 40x. Os neurónios (N) são marcados com os 22C10 marcador neuronal (vermelho, B). O ligamento (L) e tampa (C) as células são co-marcadas com anticorpos anti-αTub85E (azul, C) e um repórter específica CHO-GFP transgene (Nachman et ai, dados não publicados).

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O protocolo descrito neste vídeo oferece uma maneira de visualizar Chos proprioceptivos durante as fases de larva e pupa. Estudos sobre a estrutura eo desenvolvimento de Chos proprioceptivos têm sido até agora restrita principalmente para estágios embrionários e larvais discos imaginais. Assim, muitos aspectos de fases posteriores de desenvolvimento, de Chos tanto de larvas e adultos, são ainda desconhecidas. O protocolo descrito, em combinação com comuns ferramentas genéticas em Drosophila, pode ser utilizado para identificar e estudar novos genes e vias que desempenham um papel em fases posteriores do desenvolvimento CHOs. Este protocolo foi optimizado para a visualização de CHO, mas pode ser adaptado para a coloração de outros tecidos, tais como as asas pupa 6.

<table border="1"><tbody><tr><td> Nome do reagente / ferramenta </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Dumont # 5 (ou # 55) fórceps, biologie ponta </td><td> FST </td><td> 11252-20 ou 11252-10 (Ou pinça semelhantes) </td><td></td></tr><tr><td> Austerlitz aço inoxidável pinos de insetos, minutiens 0,1 mm </td><td> Roboz Co Instrumento cirúrgico </td><td> RS-6083-10 </td><td></td></tr><tr><td> Sylgard 184 kit elastômero de silicone </td><td> Dow Corning Corporation </td><td> 240.4019862 </td><td></td></tr><tr><td> Vannas micro tesoura (em linha reta, 7,5 cm de lâmina, 3mm) </td><td> AS Mdeizintechnik GmbH </td><td> 11-590-00 </td><td> Tesoura Primavera Vannas com especificações idênticas pode ser comprado de Roboz Cirúrgica Instrument Co. </td></tr><tr><td> Orbital - Rotamax-120 </td><td&gt; Heidolph </td><td> N / A </td><td></td></tr><tr><td> Dako Fluorescente Meio de Montagem </td><td> Dako Cytomation, Glostrup, Dinamarca </td><td> DK-5302392 </td><td></td></tr><tr><td> X10 formulação PBS </td><td colspan="2"> 2 gr / lit KCl, 2 gr / lit KH 2 PO 4, 80 gr / lit NaCl, 21,7 gr / lit Na 2 2 O HPO4.7H </td><td> A qualidade de PBS é crítica para o sucesso deste protocolo </td></tr><tr><td> PBT </td><td colspan="2"> X1 PBS + 0,1% de Tween 20 </td><td></td></tr></tbody></table>

visualization of proprioceptors in drosophila larvae and pupae

Propriocepção é a capacidade para detectar o movimento, ou posição, das partes do corpo por responder a estímulos que surgem dentro do corpo. Em drosófilas e outros insetos propriocepção é fornecida por órgãos sensoriais especializados denominados órgãos chordotonal (chos) 2. Tal como muitos outros órgãos em Drosophila, CHOS desenvolver duas vezes durante o ciclo de vida da mosca. Primeiro, os Chos larvas se desenvolvem durante a embriogênese. Em seguida, os Chos adultos começam a se desenvolver nos discos imaginais de larvas e continuar a diferenciar durante a metamorfose. O desenvolvimento de Chos larvais durante a embriogênese tem sido extensivamente estudada 10,11,13,15,16. A peça central de cada Cho é uma unidade sensorial composto de um neurônio e uma célula scolopale. A unidade sensorial é esticado entre dois tipos de células acessórias que se ligam à cutícula através de células especializadas de fixação epidérmicas 1,9,14. Quando uma mosca movimentos larva, o deslocamento relativo do ecélulas de fixação pidermal conduz ao alongamento da unidade sensorial e abertura consequente específicos transientes receptores potenciais vanilóides (TRPV) canais no segmento exterior do dendrite 8,12. O sinal de eliciada é então transferido para o circuito gerador central padrão locomotora no sistema nervoso central. CHOs múltiplas têm sido descritas na mosca adulta 7. Estes estão localizados perto das articulações dos apêndices mosca adulta (patas, asas e cabrestos) e no tórax e abdômen. Além disso, várias centenas de CHOs formam colectivamente o órgão de Johnston na antena adulto que acústico transduzir a energia mecânica 3,5,17,4. Em contraste com o amplo conhecimento sobre o desenvolvimento de CHOs em estádios de desenvolvimento embrionário, muito pouco se sabe sobre a morfologia destes órgãos durante estádios larvares. Além disso, com a excepção de CHOs femorais 18 e do órgão de Johnston, o nosso conhege sobre o desenvolvimento e estrutura de CHOs na mosca adulta é muito fragmentária. Aqui nós descrevemos um método para a coloração e visualização de CHOs em larvas de terceiro ínstar e pupas. Este método pode ser aplicado em conjunto com ferramentas genéticas para caracterizar melhor a morfologia e compreender o desenvolvimento das várias CHOs em tempo real.

Watch this Scientific Journal Video about Visualization of Proprioceptors in Drosophila Larvae and Pupae at JoVE.com

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Um método para immunostain e visualizar os órgãos chordotonal em larvas e pupas de<em&gt; Drosophila melanogaster</em&gt; É descrito.

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 ...

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

Research

JoVE Journal

Neuroscience

Visualization of Proprioceptors in Drosophila Larvae and Pupae

11.9K visualizações.  Technion-Israel Institute of Technology. Um método para immunostain e visualizar os órgãos chordotonal em larvas e pupas de Drosophila melanogaster É descrito.

Vídeo: Visualization of Proprioceptors in Drosophila Larvae and Pupae

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

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

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

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

Em células vivas, imagens de células precursoras de órgãos sensoriais na Intact Drosophila As pupas

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

Neurociência

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 ...

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 ...

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

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

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

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

Optogenetic Perturbação da atividade neural com Iluminação Laser no Semi-intacta<em&gt; Drosophila</em&gt; As larvas in Motion

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

Foraging Path-length Protocol for Drosophila melanogaster Larvae

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

Foraging Path-length Protocol for Drosophila melanogaster Larvae

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

Forrageamento Protocolo Caminho de comprimento para<em&gt; Drosophila melanogaster</em&gt; Larvas

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

Novel Assay for Cold Nociception in Drosophila Larvae

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

Novel Assay for Cold Nociception in Drosophila Larvae

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

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 ...

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 ...

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

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

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

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

Otimizando o processo de preparação de amostras para microscopia eletrônica de transmissão de junções neuromusculares em larvas de Drosophila

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

Visualização de redes de microtúbulos em junções neuromusculares e células musculares de Drosophila

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Autor em destaque: Compreendendo a rede de microtúbulos nas junções neuromusculares de Drosophila 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.