Gostaríamos de agradecer Amsale Belay por sua ajuda nas filmagens deste artigo vídeo. Este trabalho foi financiado pela CIHR, NSERC e subvenções para CRC JDL.

Parte 1. Preparação filé do abdômen Drosophila adulto. O procedimento descrito abaixo imediata prepara o abdômen para a posterior remoção da oenocytes (ver Part2 de protocolo). O mesmo procedimento também pode ser usado para a preparação de outros tecidos ligados à superfície interna da cutícula, incluindo o vaso dorsal (isto é, o coração) e gordura corporal. Note que no artigo de vídeo a técnica de dissecação oenocyte é demonstrado em um adulto de tipo selvagem voar (Canton-S) do sexo masculino, seis dias de idade. Técnicas idênticas podem ser usados ​​para remover o oenocytes de moscas fêmeas. A preparação filé abdômen leva aproximadamente 5 minutos para executar. Antes de começar é importante ter em mãos vários pinos de dissecção fina (ver abaixo e Parte 2) e uma agulha de dissecção (ver Parte 2). Pinos dissecção disponíveis comercialmente e agulhas são muitas vezes demasiado grandes para este protocolo, por isso temos levado para fabricar nosso próprio laboratório. Para criar a agulha de dissecação fio de tungstênio, (0,005 &quot;) é a primeira enfiada através do eixo da agulha hipodérmica (27G, alumínio hub). Passe o fio através da ponta da agulha hipodérmica para que emerge do hub. Crimp o fio salientes de hub;. isso impede que o fio seja puxado para fora da agulha hipodérmica Em seguida, corte o fio na extremidade oposta do tal de cravar que vários milímetros (~ 3-4mm) de fio se projetam a partir da ponta da agulha hipodérmica Passing. a ponta de tungstênio exposta através de uma chama irá aguçar o fio a um ponto muito bem. Uma pipeta de plástico descartáveis ​​5ml, quando preso no cubo da agulha hipodérmica atua como lidar com esta ferramenta conveniente para dissecção. Os pinos dissecção fina são feitas de forma semelhante caminho. Após a etapa de nitidez, segure a ponta do fio firmemente com uma pinça para evitar que se mova e fazer um corte de aproximadamente 3mm da ponta para liberar o pino de dissecção de pequeno porte. Tome cuidado para evitar que o pino de ser lançados a partir da compreensão da a pinça. Para preparar o filé de abdômen Drosophila adulto: <ol><li> Assegurar a voar para a placa de dissecção (Sylgard; Dow-Corning), colocando um pino dissecção fina que o tórax. </li><li> Remove as pernas e as asas da mosca. Ao remover o conjunto mais posterior das pernas, criar uma abertura na cutícula no ponto anterior apenas para onde o tórax do abdômen encontra. Esta abertura é usado posteriormente como um ponto de acesso para fazer uma incisão ao longo da superfície ventral do abdome (ver Etapa 5) </li><li> Garantir o abdômen da mosca no lugar, colocando um pino dissecção segundo através do segmento genital. </li><li> Cobrir a voar com Shields refrigerados e Sang inseto M3 médio (Sigma). Remover o ar, chamando-a com uma pipeta Pasteur. </li><li> Forceps usando abrir o abdómen, fazendo uma incisão ao longo da linha mediana ventral desde a abertura na cutícula criado por eliminar as pernas para o segmento genital. Retire as tripas e gônadas do abdômen. </li><li> Sever a cutícula e traquéias de ligar o abdômen para o tórax. O tecido único remanescente de ligar o abdômen para o tórax deve ser o coração. Isto proporciona a cutícula abdominal com alguma estabilidade, e impede que esta rotação ou torção durante a próxima etapa. </li><li> Pin plano da anterior maioria dos cantos da cutícula abdominal. Remover o tórax. Puxando a cutícula tenso, pin flat os cantos posterior da cutícula. O pino no segmento genital pode precisar ser reposicionado para fazer a cutícula colocar o plano. </li></ol> Parte 2. Dissecção Oenocyte A preparação filé abdômen expõe a superfície interna da cutícula abdominal e tecidos em anexo, incluindo o coração, gordura corporal, traquéia, músculos da parede do corpo, a epiderme, e os oenocytes. O coração fica ao longo da linha média dorsal. De gordura corporal (tecido opaco) cobre a maior parte da superfície interna cuticular. Traquéias (branco estruturas tubulares) se estendem desde as aberturas espiráculo lateral, e fazer ramos extensos que se infiltram nesses tecidos. Deitado debaixo destes tecidos, o oenocytes pigmentadas (de cor âmbar células) irradiam a partir da linha média para a borda lateral da cutícula na região posterior de cada segmento. O oenocytes estão diretamente sob o bronzeado regiões de cada tergito (dorsal placas cuticular) e pode ser difícil identificar, sem conhecimento prévio da sua localização. Outra população de oenocytes ventralmente localizada está associada com a sternites (ventral placas cuticular), embora seja possível isolar estas células, este protocolo de vídeo só vai demonstrar a dissecção da oenocytes dorsal. Em cada segmento abdominal, uma folha de destaque de tecido de gordura corporal está anterior à oenocytes; uma pequena folha menos óbvias de gordura corporal encontra-se posterior ao oenocytes. A folha de destaque de tecido de gordura corporal, muitas vezes cobre a oenocytes do segmento precedente. O vaso dorsal e de gordura corporal deve ser removido no primeiro fim de expor o oenocytes. É importante notar que a pigmentação amarela da oenocytes, da qual deriva o nome de seu (Oeno - do grego oinos, &quot;vinho&quot;) 1, distingue claramente estas células dos tecidos circundantes, a intensidade da pigmentação aumenta com a idade da mosca. Além disso, uma lâmina basal ensheathes a 2,3 oenocytes, dando suporte aos grupos de células e prevenindo que as células se tornem dissociada durante a dissecção. A lâmina basal também ajuda na remoção de tecidos extraneously aderido, como o tecido de gordura corporal, através da criação de um ponto fraco, onde estas aderências pode ser facilmente rompido. O procedimento descrito a seguir fornece instruções detalhadas para a remoção do oenocytes do tegumento abdominal. O oenocytes pode ser facilmente removido segmentos abdominais 2-5 (masculino) e 06/02 (feminino). O oenocytes do primeiro e último segmentos abdominal (segmentos 1 e 6 do sexo masculino ou 7 no feminino) são muitas vezes interrompidas pelos pinos dissecção segurando os cantos da cutícula e são, portanto, evitado. A remoção do oenocytes leva aproximadamente 10 minutos para executar. Para isolar o oenocytes adulto: <ol><li> Sever do tronco traqueal que emana do espiráculos usando uma agulha de dissecção. </li><li> Remover a gordura corporal e do coração, cortando as ligações à superfície interna da cutícula com uma agulha de dissecção. Para isso, posicione o ponto da agulha dissecção entre a gordura corporal ea oenocytes, e arrastá-lo a partir da borda lateral da cutícula, através da linha média, passando por baixo do coração, e continuando para o lado oposto. Progredindo do posterior ao anterior, remover o tecido de gordura corporal e do coração da cutícula de cada segmento sucessivo. Se realizada com cuidado o oenocytes permanecer solidários com a cutícula, ea gordura corporal e do coração pode ser removido todo. </li><li> Corpo de parede tira os músculos do oenocytes à superfície interna da cutícula. Utilizando a agulha de dissecção romper os músculos do corpo longitudinal parede de cada segmento. Para fazer isso, arraste a agulha de dissecção ao longo do lado posterior da vertente oenocyte, levantando-os suavemente a partir da superfície da cutícula. Os fios fita-como oenocyte deve vir longe da cutícula e podem ser estabelecidas para o lado enquanto o oenocytes restantes são removidos. </li><li> Uma vez retirado da cutícula e deitado na placa de dissecação, pode ser necessário para limpar qualquer aderido ao tecido de gordura corporal a partir do oenocytes. Isto é feito simplesmente colocando a agulha de dissecção entre o tecido de gordura corporal ea oenocytes, e cortando a ligação, empurrando a agulha para baixo e contra a placa de dissecação. </li><li> Recolher os fios oenocyte simplesmente cutucando-os com a ponta da agulha de dissecção. Uma vez preso à extremidade da agulha de dissecação do oenocytes pode ser puxado lentamente em toda a superfície do meio de cultura. O oenocytes dissecados pode ser colocado diretamente em tampão de lise celular apropriado para tanto RNA ou extração de proteínas. Note que o oenocytes (segmentos abdominais 2-5) a partir de uma mosca macho normalmente produz 15 20ng de RNA total. </li></ol> Figura Legenda: <img src="/files/ftp_upload/2242/2242fig1.jpg" alt="Figura 1" /> Figura 1. Expressão dos genes desaturase, desat1 e desatF, em oenocytes macho e fêmea de D. melanogaster. amplificação de transcrição reversa-PCR de desat1 e desatF de cDNA derivado de adultos oenocytes masculino e feminino. O gene é expresso em desat1 oenocytes tanto masculino e feminino; expressão desatF é sexualmente dimórfico, sendo expresso exclusivamente em oenocytes feminino. Correspondentes a estas diferenças na expressão gênica, desat1 é necessária para a produção de compostos de hidrocarbonetos comuns a ambos os homens e mulheres 9, e desatF está envolvido na produção de compostos de hidrocarbonetos especificamente feminino 10,11. DesatF foi mostrado para ser especificamente expressos em oenocytes feminino por em 12 de hibridização in situ. Note-se que, em média, 15 20ng de RNA total é obtido a partir da oenocytes dissecada a partir de uma mosca individual. RNA foi isolado usando o micro RNeasy kit (Qiagen); cDNA foi produzido usando o qScript síntese cDND kit (Quanta BioSciences)

<ol>
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Neste artigo de vídeo apresenta um protocolo de dissecação detalhada para a preparação do oenocytes do adulto D. melanogaster de forma adequada para a análise molecular. Um texto baseado em abreviado em conta o método de dissecação tem sido descrito em outros lugares 2, ea preparação resultando oenocyte demonstrou ser adequado para a extração de RNA e proteínas 2. Usando essa preparação também pode ser possível desenvolver métodos para a cultura de oenocytes explantado. ...

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<td>Sylgard</td>
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dissection of oenocytes from adult drosophila melanogaster

Em<em&gt; Drosophila melanogaster</em&gt;, Como em outros insetos, uma camada de cera na superfície externa da cutícula, composta principalmente de compostos de hidrocarbonetos, oferece proteção contra a dessecação e outros desafios ambientais. Vários compostos destes hidrocarbonetos cuticulares (CHC) também funcionam como sinais semiochemical, e como tal mediar comunicações pheromonal entre os membros da mesma espécie, ou em alguns casos, entre espécies diferentes, e influenciam o comportamento. Células especializadas referido como oenocytes são considerados como o principal local para a síntese de CHC. No entanto, pouco se sabe sobre o envolvimento do oenocytes na regulação da biossíntese, transporte e vias de deposição de contribuir para a saída de CHC. Dado o papel significativo que CSCs jogar em vários aspectos da biologia de insetos, incluindo a comunicação química, resistência à dessecação, e imunidade, é importante para obter uma maior compreensão da regulação molecular e genética da produção CHC dentro dessas células especializadas. O oenocytes adultos de<em&gt; D. melanogaster</em&gt; Estão localizados dentro do tegumento abdominal, e são metamerically vestida de fita-como grupos irradiando ao longo da superfície interna cuticular de cada segmento abdominal. Neste artigo vídeo que demonstrar uma técnica de dissecção utilizado para a preparação de oenocytes de adultos<em&gt; D. melanogaster</em&gt;. Especificamente, nós fornecemos uma demonstração passo a passo detalhado de (1) como filé de preparar um adulto<em&gt; Drosophila</em&gt; Abdômen, (2) como identificar as oenocytes e discerni-los de outros tecidos, e (3) como remover intacta aglomerados oenocyte do tegumento abdominal. Uma breve ilustração experimental de como esta preparação pode ser usado para examinar a expressão de genes envolvidos na síntese de hidrocarboneto é incluído. A preparação dissecados demonstrado aqui irá permitir a análise detalhada molecular e genética da função oenocyte na mosca da fruta adulto.

Watch this Scientific Journal Video about Dissection of Oenocytes from Adult Drosophila melanogaster at JoVE.com

Dissection of Oenocytes from Adult Drosophila melanogaster

Em insetos, o oenocytes produzir compostos de hidrocarbonetos cuticulares. Estes compostos protegem contra a dessecação e facilitar a comunicação química. Aqui nós demonstramos uma técnica de dissecção usado para isolar o oenocytes de adultos<em&gt; Drosophila melanogaster</em&gt;, E ilustram como esta preparação pode ser utilizada para estudar os genes envolvidos na síntese de hidrocarbonetos.

Dissecção da Oenocytes de Adultos Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

dissection-of-oenocytes-from-adult-drosophila-melanogaster

Research

JoVE Journal

Biology

Dissection of Oenocytes from Adult Drosophila melanogaster

20.3K visualizações.  University of Toronto. Em insetos, o oenocytes produzir compostos de hidrocarbonetos cuticulares. Estes compostos protegem contra a dessecação e facilitar a comunicação química. Aqui nós demonstramos uma técnica de dissecção usado para isolar o oenocytes de adultos Drosophila melanogaster, E ilustram como esta preparação pode ser utilizada para estudar os genes envolvidos na síntese de hidrocarbonetos.

Vídeo: Dissection of Oenocytes from Adult Drosophila melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissecção do CNS Larval em Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Biologia

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

Dissecação de discos imaginais de 3 larvas Drosophila Instar

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

Todo Recordings celular do cérebro de adultos Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

De alta resolução de vídeo de Rastreamento Locomotion em Adult Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

Dissecção do giro denteado do hipocampo de rato adulto

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Dissecção de Órgãos do Zebrafish Adulto

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

Isolamento de Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Injeção de insulina e Extração de hemolinfa para medir a sensibilidade à insulina em adultos Drosophila melanogaster</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Medição do Tempo de vida em<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

Dissecção e Imunocoloração do Imaginário discos de<em&gt; Drosophila melanogaster</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...