The authors wish to thank Drs. Sung-wan An and Mike O&#8217;Donnell for practical advice on establishing this assay, Dr. Chih-Jen Cheng for helpful discussions on the use of ion-specific electrodes, and Dr. Chou-Long Huang for his mentorship and support. This work was supported by the National Institutes of Health (K08DK091316 to ARR) and the American Society of Nephrology Gottschalk Award to ARR.

<ol>
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The authors have nothing to disclose.

A utilização do ensaio de Ramsay, em conjunto com eletrodos íon-específico, permite a medição das taxas de secreção de fluidos e fluxos de iões em insetos isolado Malpighi (renais) túbulos. Vinte ou mais túbulos pode ser ensaiada de cada vez, permitindo que um maior rendimento em comparação com o ensaio in vitro de indivíduo microperfused túbulos. Além disso, os eléctrodos específicos de iões permitir a determinação das concentrações de iões dentro do fluido que é segregado in situ,...

<html> Transporte de íons epitelial renal subjacente iono- organismal e osmoregulation. A Drosophila melanogaster Malpighian (renal) túbulo oferece uma oportunidade única para estudar os mecanismos moleculares de transporte de íons epitelial. Isto é devido à combinação dos poderosas da genética de Drosophila, emparelhado com a acessibilidade dos seus túbulos renais para estudo fisiológico. O ensaio de Ramsay, em homenagem ao pesquisador que foi pioneiro na técnica 1, mede as taxas de secreção de fluidos de túbulos de Malpighi isolados, e foi criada em Drosophila em 1994 pela Dow e colegas 2. Isso pavimentou o caminho para novos estudos utilizando Drosophila ferramentas genéticas, tais como o sistema GAL4-UAS 3,4, para definir específico de células vias de sinalização que regulam a secreção de fluidos. Um exemplo inclui a sinalização de cálcio, em resposta a uma hormona peptídica, 5, 6,7, entre muitos outros. ve_content &quot;&gt; uma combinação de técnicas genéticas e estudo fisiológico clássica demonstrou que a geração de urina na mosca ocorre através da secreção de um líquido rico em cloreto de potássio a partir do segmento principal do túbulo. Isto ocorre através da secreção transepitelial paralelo de catiões, principalmente K + Na +, mas também, através da célula principal, e Cl -. através da secreção de células estreladas 8-12 A capacidade de medir separadamente transepiteliais K + e Na + fluxos permite uma caracterização mais detalhada dos mecanismos de transporte do que a medição da secreção de fluido sozinho. Por exemplo, nos túbulos de Drosophila não estimuladas, a Na + / K + -ATPase ouabaína não tem efeito sobre a secreção de fluido 2, mesmo quando a sua absorção em células principais é inibida pelo inibidor de transportador aniónico taurocolato orgânica 13. No entanto, e Linton O&#39;Donnell mostrou que a ouabaína despolarizao potencial da membrana basolateral, e aumenta o fluxo de Na + 9. Como mostrado nos resultados representativos, nós replicado estes achados, e mostrou que K + fluxo é concomitantemente diminuiu 14; o aumento do fluxo de Na + e diminuição do K + fluxo têm efeitos opostos sobre a secreção de fluido, resultando em nenhuma alteração na secreção de líquido. Assim, existem duas resoluções para o &quot;paradoxo ouabaína,&quot; ou seja, a observação inicial de que a ouabaína não tem efeito sobre a secreção de fluido no túbulo Drosophila:. Em primeiro lugar, nos túbulos estimulada, o efeito de ouabaína na secreção de fluido não é aparente devido à sua absorção pelo transportador de aniões orgânicos 13; e, segundo, nos túbulos não estimuladas, ouabaína tem efeitos sobre transepithelial Na + e K + fluxo opostas, resultando em nenhuma alteração líquida na secreção de fluidos (ver resultados representativos e ref. 9). Portanto, o principal papel do Na + / K + -ATPase nos túbulos não estimuladas é o de diminuir a concentração intracelular de Na + para gerar um gradiente de concentração de Na + para o favorável -coupled processos de transporte através da membrana basolateral. Com efeito, medindo em separado de Na + e K + fluxos, nós demonstramos que túbulos que falta o co-transportador de mosca de sódio-potássio-2-cloreto de (CCAN) diminuíram K transepitelial + fluxo, sem diminuição adicional após a adição de ouabaína, e nenhuma alteração em transepitelial Na + fluxo 14. Estes resultados suportada nossa conclusão de que o Na + que entra na célula através da CCAN é reciclado através do Na + / K + -ATPase. Em outro exemplo, Ianowski et al., Observaram que a redução da concentração do banho de K + de 10 mM a 6 mM de K + diminuiu transepitelial fluxo transepitelial e aumentou o fluxo de Na + nos túbulos de Rhodnius prolixus, sem alteração líquida no fluido de secreção <sup&gt; 15. Efeitos diferenciais de fluxo de Na + e K + fluxo através dos túbulos de larvas também têm sido observadas em túbulos de Drosophila em resposta a diferentes dietas de sal 16 e em duas espécies de mosquitos em resposta à criação de salinidade 17. O maior desafio na medição de fluxo de iões transepitelial na preparação de ensaio Ramsay é a determinação das concentrações de iões dentro do fluido segregado. Este desafio foi cumprido com soluções diferentes, incluindo photometery chama 18, o uso de íons radioativos 19, e eletrônica sonda comprimento de onda espectroscopia por dispersão de 20. Estas técnicas exigem transferência da gota líquido secretado a um instrumento para a medição de concentrações de íons. Uma vez que o volume de fluido que é segregado pelo túbulo Drosophila não estimulado é pequena, tipicamente ~ 0,5 nl / min, isto representa um desafio técnico e também introduz erro se algum do fluido é segregadaperdeu por ocasião da transferência. Em contraste, a utilização de eléctrodos específicos de iões permite a medição da actividade de iões (a partir do qual a concentração de iões pode ser calculado) in situ. O protocolo corrente foi adaptada da utilizada por Maddrell e colegas para medir transepitelial K + fluxo através do túbulo Rhodnius usando valinomicina como o K + ionóforo 21, e também descreve a utilização de um -butylcalix 4- terc [4] areno-ácido tetraacético tetraetil éster à base de Na + eléctrodo específico de iões especï¿½ico caracterizado por Messerli et. al. 22. Eletrodos íon-específicas também têm sido usados ​​para medir concentrações de íons no líquido secretado por túbulos de Malpighi no ensaio Ramsay em 9,23 adulto e 16 larval Drosophila melanogaster, a Nova Zelândia Alpine Weta (Hemideina maori) 24 e 17 em mosquitos. Aqui, descrevemos em detalhes o uso do Ramsay comodizer para medir as taxas de secreção de fluidos em túbulos de Malpighi de Drosophila melanogaster, bem como o uso de eletrodos de íon-específico para determinar as concentrações de K + e Na + dentro do fluido secretado e, portanto, o cálculo dos fluxos de iões transepithelial. Uma visão geral do ensaio é fornecida na Figura 1. <img alt="figura 1" src="/files/ftp_upload/53144/53144fig1.jpg" /> Figura 1. Esquema do Malpighian Palhinha eo Ramsay Ensaio com uso de íon-específicas eletrodos para medir concentrações de iões. Esta figura ilustra a configuração para o ensaio Ramsay. (A) Cada mosca tem quatro túbulos, um par de túbulos anterior e um par de túbulos posteriores, que flutuam na cavidade abdominal cercado por hemolinfa. Em cada par, as duas túbulos juntar-se no ureter, que, em seguida, esvazia-se a urina na junção do intestino médio e hindgut. Os túbulos são cegos-ended. A urina é gerado pelo segmento principal secretor de fluido (representado em vermelho), e flui para fora do ureter e no intestino. Após dissecção, a par dos túbulos é dissociada do intestino, ao seccionar o ureter. (B) A par dos túbulos é então transferida para uma gota de solução salina de banho dentro de uma cavidade do prato de ensaio. Um dos dois túbulos, referido aqui como o &quot;túbulo âncora&quot;, é enrolado em torno de um pino de metal e é inerte. A outra é o túbulo túbulo secretora. O segmento inicial (que não secretam fluido) e segmento principal do túbulo segregam permanecer dentro da gotícula de solução salina de banho. Íons e movimento de água da salina de banho e para o lúmen do túbulo do segmento principal, em seguida, avançar em direção ao ureter, como ocorreria in vivo. O segmento inferior (azul) está fora da salina de banho e, portanto, inertes. Uma vez que o ureter é cortado, o fluido segregado surge como uma gota a partir da extremidade de corte do ureter. Tele secretada gota de fluido aumenta ao longo do tempo como a secreção continua, e o seu diâmetro é medido usando um micrómetro ocular. Uma camada de óleo mineral evita a evaporação do fluido que é segregado. Os eletrodos de referência específicas e de iões de medir a concentração de íons do fluido secretado. <a href="https://www.jove.com/files/ftp_upload/53144/53144fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Ellsworth Adhesives</td><td>http://www.ellsworth.com/dow-corning-sylgard-184-silicone-encapsulant-0-5kg-kit-clear/</td><td>May be purchased from multiple distributors</td></tr><tr><td>Petri dish, polystyrene, 100 mm x 15 mm</td><td>Fisher</td><td>FB0875712</td><td>Specific brand is not important</td></tr><tr><td>Petri dish, polystyrene, 35 mm x 10 mm</td><td>Corning Life Sciences</td><td>Fisher 08-757-100A</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Blades #1</td><td>Fine Science Tools</td><td>10011-00</td><td>Specific brand is not important; use appropriate sharps precautions</td></tr><tr><td>Needle, 30 G x 1/2</td><td>Becton Dickinson</td><td>305106</td><td>Use appropriate sharps precautions</td></tr><tr><td>Minutien pins, black anodized, 0.15 mm</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Stereomicroscope with ocular micrometer</td><td>Nikon</td><td>SMZ800</td><td>Specific brand is not important; this is given as an example</td></tr><tr><td>Sheet of black stained glass, 3 mm (1/8 inch) thick</td><td>Hobby shop</td><td></td><td>Example includes Spectrum Black Opal by Spectrum Glass (http://www.delphiglass.com/spectrum-glass/opalescent/spectrum-black-opal)</td></tr><tr><td>Glass cutting tools (glass cutter, glass cutting pliers)</td><td>Hobby shop</td><td></td><td>Examples include the Studio Pro Lightweight Running Pliers by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/pliers-nippers/studio-pro-lightweight-running-pliers) and the Studio Pro Brass Glass Cutter by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/glass-cutters/studio-pro-brass-glass-cutter). Use appropriate safety precautions when cutting glass</td></tr><tr><td>Borosilicate glass capillary tube, unfilamented, GC120-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0042</td><td></td></tr><tr><td>Borosilicate glass capillary tube, filamented, GC120F-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0044</td><td></td></tr><tr><td>Nitric acid, 70%</td><td>Sigma</td><td>438073</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines. Specific brand is not important</td></tr><tr><td>Cimarec 7 in x 7 in hotplate</td><td>Fisher</td><td>11675911Q</td><td>Specific brand is not important; caution when heated</td></tr><tr><td>Selectophore dichlorodimethylsilane</td><td>Sigma</td><td>40136-1ML</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Two-step vertical pipet puller</td><td>Narishige</td><td>PC-10</td><td>Other pipet pullers can be used; this is given as an example</td></tr><tr><td>Glass petri dish, 150 mm diameter x 15 mm height</td><td>Fisher</td><td>08-748E</td><td>Specific brand is not important; only one dish needed</td></tr><tr><td>World Precision Instruments E210 1 mm micropipette storage jar</td><td>Fisher</td><td>50-821-852</td><td>May be available from other distributors. Useful to have two jars. Note that although this jar is specified for 1 mm pipets, and the pipets used here are 1.2 mm, in our experience the 1 mm jar works best for the 1.2 mm pipets.</td></tr><tr><td>Silica Gel, Tel-Tale Desiccant, indicating, 10-18 mesh</td><td>Fisher</td><td>S161-500</td><td>Indicating silica useful for determining whether silica gel retains desiccating ability</td></tr><tr><td>World Precision Instruments MicroFil, 34G</td><td>Fisher</td><td>50-821-914</td><td>May be available from other distributors.</td></tr><tr><td>1 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309659</td><td>May be available from other distributors.</td></tr><tr><td>3 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309657</td><td>May be available from other distributors.</td></tr><tr><td>D300 3-way stopcock with female luer lock inlet port, male luer outlet port with rotating collar and guard</td><td>Cole-Parmer</td><td>UX-30600-02</td><td>Specific brand is not important</td></tr><tr><td>Female Luer Locking Connector</td><td>4 Medical Solutions</td><td>ADC 9873-10</td><td>Specific brand is not important; barbed end is ~4 mm at narrowest point and ~7 mm at widest point.</td></tr><tr><td>Silicone Tubing I.D. x O.D. x Wall: 1/16 x 1/8 x 1/32 in. (1.59 x 3.18 x 0.79 mm)</td><td>Fisher</td><td>14-179-110</td><td>Specific brand is not important</td></tr><tr><td>E-3603 tubing, I.D. x O.D.: 1/32 x 3/32 in</td><td>Fisher</td><td>14171208</td><td>Specific brand is not important</td></tr><tr><td>Modeling clay</td><td></td><td></td><td>Specific brand is not important</td></tr><tr><td>Selectophore potassium ionophore I, cocktail B</td><td>Sigma</td><td>99373</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Selectophore sodium ionophore X</td><td>Sigma</td><td>71747</td><td>Sodium ionophore X = 4-tert-butylcalix[4]arene-tetraacetic acid tetraethylester</td></tr><tr><td>Selectophore 2-nitrophenyl octyl ether</td><td>Sigma</td><td>73732</td><td></td></tr><tr><td>Selectophore sodium tetraphenylborate</td><td>Sigma</td><td>72018</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Schneider&#039;s Drosophila medium</td><td>Life Technologies</td><td>21720024</td><td></td></tr><tr><td>High impedance electrometer</td><td>World Precision Instruments</td><td>FD223a</td><td></td></tr><tr><td>Microelectrode holder 1 mm with 45&amp;deg; body, vented, with handle</td><td>Warner Instruments</td><td>64-1051</td><td></td></tr><tr><td>Microelectrode holder 1 mm with straight body, vented</td><td>Warner Instruments</td><td>64-1007</td><td></td></tr><tr><td>Silver wire</td><td>Warner Instruments</td><td>64-1318</td><td></td></tr><tr><td>Micromanipulators, pair</td><td>Leitz</td><td></td><td>Various brands/models will work; this is an example</td></tr><tr><td>Faraday cage</td><td>Technical Manufacturing Corporation</td><td>81-334-03</td><td>This is an example; any Faraday cage will work</td></tr><tr><td>Single gooseneck fiberoptic light</td><td>Nikon</td><td></td><td>Specific brand is not important</td></tr><tr><td>mineral oil</td><td>Fisher</td><td>BP-2629</td><td>Specific brand is not important</td></tr><tr><td>forceps, Dumont #5 with Biologie tip</td><td>Fine Science Tool</td><td>11295-10</td><td>May be available from other distributors.</td></tr></tbody></table>

use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule

Modulação de transporte iónico epitelial renal permite que os organismos para manter a homeostase iónica e osmótica na face da variação das condições externas. A Drosophila melanogaster Malpighian (renal) túbulo oferece uma oportunidade única para estudar os mecanismos moleculares de transporte de íons epitelial, devido à genética poderosas deste organismo e da acessibilidade dos seus túbulos renais para estudo fisiológico. Aqui, nós descrevemos o uso do ensaio de Ramsay para medir as taxas de secreção de fluidos a partir de isolado de moscas túbulos renais, com a utilização de eléctrodos específicos de iões para medir as concentrações de sódio e potássio em que o fluido segregado. Este ensaio permite o estudo de fluidos e de iões transepitelial fluxos de ~ 20 túbulos de cada vez, sem a necessidade de transferir o fluido segregado a um aparelho separado para medir as concentrações de iões. Túbulos geneticamente distintas podem ser analisados ​​para avaliar o papel de genes específicos em processos de transporte. Além disso, o bathing salina pode ser modificado para analisar os efeitos das suas características químicas, ou drogas ou hormonas acrescentados. Em resumo, esta técnica permite a caracterização molecular dos mecanismos básicos de transporte epitelial de iões no túbulo Drosophila, bem como a regulação destes mecanismos de transporte.

As Figuras 7 e 8 demonstram que a utilização do ensaio de Ramsay com eléctrodos específicos de iões para medir as concentrações de K + e Na + pode distinguir geneticamente e farmacologicamente distintas K + e fluxos de Na +, a informação que não é capturada medindo as taxas de secreção de fluidos sozinho. Figura 7 mostra que a diminuição da secreção de fluido nos túbulos de moscas que transportam uma mutação nula homozigót...

Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Este protocolo descreve a utilização do ensaio de Ramsay para medir as taxas de secreção de fluidos de Malpighi (túbulos renais) isolado a partir de Drosophila melanogaster. Além disso, a utilização de eléctrodos específicos de iões para medir as concentrações de sódio e potássio no líquido segregado, que permite o cálculo do fluxo de iões transepitelial, é descrito.

A utilização do Ramsay ensaio para medir secreção de fluidos e Ion Flux Preços no<em&gt; Drosophila melanogaster</em&gt; Tubule Malpighian

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

use-ramsay-assay-to-measure-fluid-secretion-ion-flux-rates-drosophila

Research

JoVE Journal

Biology

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

11.7K visualizações.  University of Texas Southwestern Medical Center.  Este protocolo descreve a utilização do ensaio de Ramsay para medir as taxas de secreção de fluidos de Malpighi (túbulos renais) isolado a partir de Drosophila melanogaster. Além disso, a utilização de eléctrodos específicos de iões para medir as concentrações de sódio e potássio no líquido segregado, que permite o cálculo do fluxo de iões transepitelial, é descrito. 

Vídeo: Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

Ensaio para a integridade da barreira hematoencefálica na Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Biologia do Desenvolvimento

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Medindo respostas fisiológicas de<em&gt; Drosophila</em&gt; Neurônios sensoriais para Lipid feromonas Usando Imagem de cálcio ao vivo

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Imunologia e Infecção

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

O capilar alimentador ensaio mede ingestão alimentar em<em&gt; Drosophila melanogaster</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Neurociência

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

Quantificação da ingestão de Macronutrientes em uma Tela Neuronal Termogenética usando Larvas de Drosophila

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Medição da Actividade larval no<em&gt; Drosophila</em&gt; Monitor de Atividade

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Biologia

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

PH intracelular de quantificação de óptica em Drosophila melanogaster túbulo de Malpighi epitélios com um indicador de pH fluorescente geneticamente codificado

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

Estudando a Atividade de Neuropeptídeos e Outros Reguladores do Sistema Excretório no Mosquito Adulto

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...

Monitoring Gut Acidification in the Adult Drosophila Intestine

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the proper functioning of the gut. One such region, the copper cell region (CCR), is localized to the middle midgut and consists, in part, of a group of cells known as copper cells. Copper cells are involved in gastric acid secretion, an evolutionarily conserved process whose precise role is poorly understood. This paper describes improvements in the current protocol used to assay for acidification of the adult Drosophila melanogaster gut and demonstrates that it can be used on other species of flies. In particular, this paper demonstrates that gut acidification is dependent on the fly's nutritional status and presents a protocol based on this new finding. Overall, this protocol demonstrates the potential usefulness of studying Drosophila copper cells to uncover general principles underlying the mechanisms of gut acidification.

Monitoring Gut Acidification in the Adult Drosophila Intestine

Here, we present a standardized protocol for monitoring gut acidification in Drosophila melanogaster with optimal output. We first use this protocol for gut acidification monitoring in Drosophila melanogaster and then demonstrate its use in non-model Drosophila species.

Monitoramento da acidificação intestinal no intestino de Drosophila adulto

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the ...

Drosófila Análise de depósito fecal para descoberta de medicamentos

Assessment of The Effect of Antidiarrheal Drugs and Plant Extracts on Drosophila melanogaster

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a model to study intestinal function, only a few reports have focused on Drosophila melanogaster (D. melanogaster). Compared to mice, fruit flies present many advantages, such as a short life cycle, cost-effective and simple maintenance, and no ethical issues. Furthermore, the mammalian gastrointestinal physiology, anatomy, and signaling pathways are highly conserved in D. melanogaster. Plant extracts have been used traditionally to treat diarrhea and constipation. For example, Psidium guajava (P. guajava) is one of the most known antidiarrheal agents in the tropics. However, no studies have evaluated the effect of antidiarrheal and laxative drugs and plant extracts in D. melanogaster, and it remains unknown if similar effects (e.g., smaller, more concentrated, and less abundant fecal deposits in the case of antidiarrheal drugs) can occur in the fruit flies compared to mammals. In this study, an antidiarrheal effect induced by P. guajava is demonstrated in a D. melanogaster strain that presents a diarrheic phenotype. Fecal sampling produced by flies is monitored using a dye-supplemented food. This protocol outlines the method used for preparing food with drugs, evaluating the fecal deposits of flies fed on these food preparations, and interpreting the data obtained.

Autor em destaque: Avançando na pesquisa antidiarreica com o modelo de Drosophila 

Here, a method is described to feed Drosophila melanogaster with drugs and plant extracts and assess their effect on the gastrointestinal tract by analyzing the fruit flies fecal deposits. The drug-treated flies can be used as a model for further research.

Avaliação do Efeito de Drogas Antidiarreicas e Extratos Vegetais sobre Drosophila melanogaster

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a ...

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