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1. Manutenção de Gaiolas População <ol><li> Manter gaiolas população a 25 ° C num ciclo de luz-escuro de 12 h. Enquanto os grupos de controlo e experimentais estão expostas às mesmas condições de iluminação, esta técnica pode ser realizada em ambiente de laboratório padrão. </li><li> Permitir fêmeas põem ovos durante a noite em placas de ágar-suco de maçã. </li><li> Recolha de larva, mantendo as placas com os ovos depositados recentemente, a 25 ° C durante 24 hr. Colocar uma pequena dose de levedura no meio da chapa para atrair as larvas eclodidas. </li><li> Recolhe 1 r larvas que migraram para a pasta de levedura, no centro da placa de sumo de maçã. Use uma espátula de metal para transferi-lo para uma placa de suco de maçã fresca. Pasta de levedura adicional pode ser adicionado para garantir que não é uma alimentação adequada até que os ensaios são realizados. Placas de sumo de uva, também pode ser usado no lugar de placas de sumo de maçã. </li><li> Obter tarde 2 ª e início 3 º instar larvae permitindo 1 º ínstar de idade para 40-48 horas. Dentro desta faixa das taxas de alimentação são constantes 13. A idade pode ser confirmado por meio de exame dos ganchos na boca uma vez que existem alterações distintas na estrutura desta com cada um muda de larvas. </li><li> Tarde 2 larvas 3 ª nd-precoce são coletados para ensaios de comportamento suavemente e extensivamente lavando pratos suco de maçã com água e coleta de larvas em um filtro de malha. As larvas são então transferidos para uma placa de ágar. </li><li> Todas as análises são realizadas em paralelo com o controlo e animais experimentais. </li></ol> 2. Paradigma Comportamental - Locomotion <ol><li> Colocar uma única larva de instar 3 rd sobre um substrato de agar a 2% em 100 milímetros de cultura de tecidos de prato e permitir a larva para aclimatar-se durante 30 seg. Larvas usar seus sclerites cefalofaringeano (ganchos boca) para impulsionar seus corpos em toda a superfície do substrato de ágar. Isso é feito em um prato separado, porque eut é mais difícil de visualizar as contracções do corpo na solução de levedura como o animal é quase a mesma cor que a levedura. </li><li> Observar e registrar cada posterior ao movimento anterior sobre o substrato por um período de 1 min. n = 20 para cada genótipo. Não mais do que 10 animais deverão ser analisadas por placa. </li></ol> 3. Comportamento Paradigma - Alimentação <ol><li> Usando rombas Inox # 5 pinças, transferir cuidadosamente o estádio de larva 3 rd da placa de agar locomotora para o centro de uma placa de ágar-cheia coberto com 5 ml de solução de 2% de levedura de padeiro activado. Certifique-se de que a solução é homogênea como o fermento vai resolver com o tempo. Quando na solução de levedura, as larvas em grande parte permanecem no local e alimentação, facilitando a observação do comportamento. A taxa de contracções gancho boca correlaciona-se directamente com a quantidade de alimento ingerido 14. </li><li> Permitir larva para se aclimatar para 30 seg. </li><li> Observar e registrar o número decontrações gancho na boca por um período de 1 min. n = 20 para cada genótipo. </li></ol> 4. Dissecções Gut larval <ol><li> Faça uma solução fixação em formol EM-grau 4% em tampão fosfato 1x (PBS) e coloque em um copo ponto 3 poços. </li><li> Dissecar cuidadosamente vagando tarde 3 º instar coragem de larvas em um prato de vidro 3-bem com solução PBS 1x, certificando-se de cada vez que o proventrículo é deixada intacta. Com uma pinça, segure a extremidade posterior, e com a outra, segure os ganchos na boca. Enquanto segura o imóvel extremidade posterior, puxe delicadamente sobre os ganchos na boca para ter acesso a coragem. Remova todos os tecidos associados (glândulas salivares, cérebro, corpo de gordura, etc), em seguida, transferir cada intestino para o prato 3 poços contendo a correção formaldeído. Vagando 3 º instar larva são usados ​​porque nesta fase de desenvolvimento, a larva deixa de se alimentar em preparação para pupariation, eo proventrículo é limpo de levedura. </li> &lt;li&gt; Incubar entranhas a 4 ° C durante a noite numa caixa de cultura de tecido opaco. </li><li> Antes de retirar solução fixação em formol a partir de poços, remova o ceco gástrico e prenda o intestino médio ~ 150 ï ¿½ 2 do proventrículo de modo que as projeções podem ser claramente vistos sem impedimento. </li><li> Retirar correcção de formaldeído a partir de poços e substituir com 1x PBT (1x PBS, 0,1% livre de protease de albumina de soro bovino, X-100 0,1% de Triton) solução tampão. Lave coragem 6x por 10 min em 1x PBT. Coloque amostras de tecido em um rotor mecânico ao fazer lavagens. </li><li> Incubar a 4 ° C durante 1 hora em 10 -6 M de 5-HT para melhorar a sinalização da serotonina. Lave coragem 6x por 10 min em 1x PBT. Estudos anteriores demonstraram que esta concentração de exógena 5-HT não afeta a arquitetura neuronal ou densidade varicosity em análises de imuno-histoquímica e simplesmente aumenta o sinal de ruído 15-16. </li><li> Incubar a 4 &amp;# 176; C durante a noite em anticorpo primário anti-serotonina (monoclonal criado em mouse ou policlonal em coelho). Lave coragem 6x por 10 min em 1x PBT. Coloque amostras de tecido em um rotor mecânico ao fazer lavagens. </li><li> Incubar a 4 ° C durante 90 min, em anticorpo secundário (Alexa Fluor 568 anticorpo de cabra anti-ratinho ou anti-IgG de coelho, diluição 1:400). Lave coragem 6x por 10 min em 1x PBT. Coloque amostras de tecido em um rotor mecânico ao fazer lavagens. </li><li> Incubar em carbonato de sódio 4 mM durante 10 min num rotor mecânico, em seguida, montar em 4% de n-propilo gallate/20 mM de carbonato de sódio, e vista sob fluorescência. O carbonato de sódio é utilizada para converter as amostras para o tampão e o pH utilizado nos meios de montagem. </li><li> Capturar imagens de amostras de tecidos com a ampliação de 400X imunocoradas para análise. </li></ol> 5. Análise de Neural dos circuitos <ol><li> Fibras de neurites foi quantificado (número e tamanho das varizese grau de ramificação) usando Neuroleucida e Neuroexplorer. No entanto, isso também pode ser feito manualmente ou utilizando o Simple Neurite Tracer (um aplicativo gratuito que pode ser baixado online). </li><li> Trace as projeções de fibra individuais do cérebro para o proventrículo e quantificar número varicosity, ramos e do número de grandes varizes por unidade de comprimento. As fibras axonais projetando a partir do cérebro são empacotados no nervo recurrens e não são acessíveis para análise até atingirem o proventrículo, onde eles se separam e fasciculada. </li></ol>

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Não temos nada a divulgar.

Desenvolvimento aberrante do circuito stomatogastric serotoninérgica, que ocorre durante o final de embriogênese, afetará a sua função madura. As alterações na arquitectura de neurites que inervam o intestino pode ser correlacionada com a saída funcional do circuito, que é a taxa de alimentação (medido por contracções gancho boca numa solução de levedura) (Figura 1). O uso do sistema bipartido UAS-Gal4 em Drosophila torna possível visar especificamente para cima ou para baixo re...

Drosophila é um sistema modelo extremamente poderosa para o estudo do desenvolvimento neural circuito devido ao tempo de geração rápidos e de baixo custo experimental, e a capacidade de manipular e controlar os factores genéticos e ambientais. Neurogênese, descoberta caminho neuronal e sinaptogênese são conservados entre os humanos e Drosophila, portanto os mecanismos de criação, manutenção e modificação de circuitos neurais são conservadas também. Neurotransmissores clássicos, como a serotonina (5-hidroxitriptamina ou 5-HT) podem servir como fatores de crescimento antes de adotar seus papéis como moléculas de sinalização no circuito neural maduro 1-3 Estudos anteriores demonstraram que os níveis perturbado de 5-HT durante o desenvolvimento embrionário altera a conectividade de neurónios maduros 4. Outros mostraram que a aplicação ectópica de 5-HT dos neurónios Helisoma reprimir o crescimento de neurites em cultura, bem como sinaptogénese 5-7. Em Drosophila, desenvolvimento níveis de 5-HT está inversamente relacionada com o número e tamanho varicosidade, bem como o grau de arborização urbana, ao longo do comprimento de neurites que se projectam para o intestino anterior do SNC 8. Neurotransmissão serotonérgica foi mostrado para modular comportamentos alimentares em diversas espécies, incluindo Drosophila 8-9. O circuito de alimentação em Drosophila é um circuito relativamente simples que pode ser usado como um modelo para correlacionar a saída funcional (alimentação) com alterações no desenvolvimento das projecções axonais do cérebro para o intestino anterior. Schoofs et al. têm mostrado que a Drosophila alimentação larval é regulada por geradores de padrão central que influenciam a musculatura 10. Embora a anatomia muscular específico não é completamente compreendido, tem sido mostrado que o nervo da antena, nervo maxilar, e nervo acessório protorácico são responsáveis ​​pelas metas musculares envolvidos nocomportamento alimentar. A maioria dos dados que envolvem a musculatura e nervo anatomia de alimentação de invertebrados é limitado a larvas Calliphora. A taxa de alimentação de larvas de segundo instar pode ser avaliada pela retração dos sclerites cefalofaringeano (ganchos na boca), e é reprodutível e de alto rendimento. As placas cefalofaringeano são inervados por fibras do centro de neurônios de 5-HT através do nervo frontal. O proventrículo, ou intestino anterior, é inervado por fibras serotoninérgicos (recurrens nervo) que fasciculada no intestino médio e são responsáveis ​​por contrações do intestino anterior (Figura 1) 11-12. Alterações na ramificação axonal, e o número e tamanho das varizes ao longo do comprimento da neurite, pode ser quantificada utilizando técnicas de imuno-histoquímica. Manipulando neuronal da 5-HT durante o desenvolvimento, quer directa quer indirectamente, pode alterar a saída funcional do circuito de alimentação, que pode ser avaliada e correlacionadas com as mudanças na morphology da arquitetura neurite.

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:217px;">Eclipse E-800 Microscope</td>
			<td style="width:175px;">Nikon Instruments</td>
			<td style="width:115px;"></td>
			<td style="width:251px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:217px;">Neuroleucida</td>
			<td style="width:175px;">MBF Biosciences</td>
			<td style="width:115px;">NL-15</td>
			<td style="width:251px;">Used to analyze gut fiber architecture, not necessary to have</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:217px;">Northern Eclipse</td>
			<td style="width:175px;">Empix Inc</td>
			<td style="width:115px;"></td>
			<td style="width:251px;">Imaging software</td>
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		<tr height="60">
			<td height="60" style="height:60px;width:217px;">G-2E/C TRITC EX 528-553</td>
			<td style="width:175px;">Nikon Instruments</td>
			<td style="width:115px;">96312</td>
			<td style="width:251px;">Filter for specific secondary antibody</td>
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		<tr height="80">
			<td height="80" style="height:80px;width:217px;">N.A. 0.75; W.D. 0.72 mm; DIC Prism: 40xI, 40x I-C; Spring loaded</td>
			<td style="width:175px;">Nikon Instruments</td>
			<td style="width:115px;">MRH00400</td>
			<td style="width:251px;">Objective used for imaging</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:217px;">Simple Neurite Tracer</td>
			<td style="width:175px;">NIH Image J</td>
			<td style="width:115px;"></td>
			<td style="width:251px;"><a href="http://fiji.sc/Simple_Neurite_Tracer">http://fiji.sc/Simple_Neurite_Tracer</a></td>
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functional analysis of the larval feeding circuit in drosophila

O circuito de alimentação serotonérgica em larvas de Drosophila melanogaster pode ser usado para investigar substratos neuronais de importância crítica durante o desenvolvimento do circuito. Usando a saída funcional do circuito de alimentação, alterações na arquitectura neuronal do sistema estomatogástrico pode ser visualizado. Comportamento alimentar pode ser registada por meio da observação da taxa de retracção dos ganchos na boca, que recebem a inervação do cérebro. Comportamento locomotor é usado como um controlo fisiológico da alimentação, uma vez que as suas larvas usar ganchos na boca para percorrer através de um substrato de agar. Alterações no comportamento alimentar pode ser correlacionada com a arquitectura axonal das neurites que inervam o intestino. Usando imunohistoquímica é possível visualizar e quantificar essas mudanças. O manuseio inadequado das larvas durante paradigmas de comportamento pode alterar dados como eles são muito sensíveis a manipulações. Imagem adequada da arquitetura neurite inervaçãoo intestino é crítica para a quantificação exacta do número e tamanho das varizes, bem como o grau de nós de ramificação. Análise da maioria dos circuitos de permitir apenas a visualização da arquitetura neurite ou efeitos comportamentais, no entanto, este modelo permite correlacionar a saída funcional do circuito com as deficiências na arquitetura neuronal.

O circuito de alimentação serotoninérgica no larva Drosophila pode servir como um modelo extremamente eficaz para observar a influência de fatores específicos no desenvolvimento do sistema nervoso. Quantificando taxa de alimentação, é possível ligar a arquitectura axonal do circuito de alimentação, com a sua saída funcional (Figura 1). O ensaio locomotor é usado como um controlo fisiológico para as retracções dos ganchos na boca, uma vez que as suas larvas usar ganchos na boca p...

Watch this Scientific Journal Video about Functional Analysis of the Larval Feeding Circuit in Drosophila at JoVE.com

Functional Analysis of the Larval Feeding Circuit in Drosophila

O circuito de alimentação em larvas de Drosophila melanogaster serve um modelo simples, mas poderosa que permite mudanças na taxa de alimentação a ser correlacionada com alterações no circuito neural stomatogastric. Este circuito é composto por neurónios serotonérgicos centrais que enviam projecções para os ganchos na boca, bem como o tubo digestivo anterior.

Análise Funcional do circuito de alimentação das larvas em Drosophila</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

functional-analysis-of-the-larval-feeding-circuit-in-drosophila

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JoVE Journal

Neuroscience

Functional Analysis of the Larval Feeding Circuit in Drosophila

10.2K visualizações.  Saint Louis University School of Medicine.  O circuito de alimentação em larvas de Drosophila melanogaster serve um modelo simples, mas poderosa que permite mudanças na taxa de alimentação a ser correlacionada com alterações no circuito neural stomatogastric. Este circuito é composto por neurónios serotonérgicos centrais que enviam projecções para os ganchos na boca, bem como o tubo digestivo anterior. 

Vídeo: Functional Analysis of the Larval Feeding Circuit in Drosophila

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

Visualização de Larval Nervos segmentar em 3 Rd Instar Drosophila Preparações larval

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

Neurociência

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Em Visualization vivo das vesículas sinápticas Dentro Drosophila larval Axônios segmentar

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Análise morfológica de Drosophila Larval sensorial periférica Dendrites Neuron e axônios Usando Mosaicos Genéticos

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

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

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

Dissecção e imunohistoquímica de larvas, pupa e adulto<em&gt; Drosophila</em&gt; Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Monte todo imunomarcação de Neurônios Receptores Olfatórios no<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Imagens ao vivo de<em&gt; Drosophila</em&gt; larval Neuroblastos

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Por Quantificação Matérias: Caracterização de fenótipos na<em&gt; Drosophila</em&gt; Larval Junção Neuromuscular

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Remoção de<em&gt; Drosophila</em&gt; Tecido muscular larval Filetes para Análise de imunofluorescência de neurônios sensoriais e células epidérmicas

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Macropatch focal gravações das correntes sinápticas da junção Neuromuscular Larval da drosófila

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...