Agradecemos a RIKEN para financiamento. P10-Gal4 foi um presente tipo de Alain Vincent (Université Paul Sabatier, Toulouse, França).

1. Preparação de reagentes Notas antes de começar: dissecção e coloração imuno-histoquímica são realizadas em uma câmara magnética ea larva é preso utilizando os pinos de insetos com formato especial. Instruções detalhadas sobre a construção de uma câmara magnética, ea preparação desses pinos pode ser encontrada nas referências relacionadas 14,15. Em resumo, um buraco quadrado 1x1cm é cortada em uma folha magnética e uma lamela afixada na parte traseira da folha para fazer uma pequena câmara. Os lados da câmara são selados com cola epóxi, após esta cola não definiu a câmara é lavado várias vezes com etanol 70% antes de usar. Pinos de insetos dissecção são preparados por flexão na forma requerida e então colados em uma guia de metal 14,15. Em alternativa a uma guia de metal, temos utilizado uma invertida de cabeça chata pin desenho de aço com uma alça feita a partir de uma ponta de corte amarela. O uso deste arranjo câmara magnética permite fechar controle over posicionamento do pino e do tecido que se estende durante a dissecção. Para dirigir a expressão do gene repórter em subconjuntos diferentes de investigadores DA neurônios pode usar várias linhas diferentes Gal4 (resumido por Shimono e colegas 16). Muitas dessas linhas estão disponíveis em centros de estoque público. Neste protocolo representante, realizamos imunocoloração de uma linha em que duas classes contrastantes de DA neurônio são co-marcado: o mais simples de classe I e mais complexo de classe IV (P10-Gal4 17,18, UAS-mCD8:: Kusabira-Orange (KO)). <ol><li> Prepare Ca + + livre HL3.1 salina <ol><li> Em mM: 70 NaCl, KCl 5, 20 MgCl 2, 10 NaHCO 3, 5 HEPES, 115 sacarose, trealose e 5; pH 7,2 19. Filtro-esterilizar e armazenar a 4 ° C. Nota: Ca + + livre solução impede a contração muscular durante a dissecção. </li></ol></li><li> Prepare tampão PHEM 2x <ol><li> Em mM: 130 PIPES, HEPES 60, 20 EGTA, 4 MgSO4; pH 7,0. Filtro-esterilizar e armazenar a 4 ° C. Nota: Estes materiais não se dissolvem até que a aproximação do pH 7.0. </li></ol></li><li> Prepare o fixador recém imediatamente antes da fixação. <ol><li> A fim de preparar uma solução de 25ml, paraformaldeído 2g primeiro mix, 100μl 1M NaOH e água 10ml em tubo Falcon 50ml. </li><li> Agite fixador em um banho de água 55 ° C, com agitador até que a solução é clara. (Volume total será de cerca de 11,5 ml.) </li><li> Fixador fresco no gelo. </li><li> Adicionar 12.5ml tampão PHEM 2x. </li><li> Ajustar o pH para 7,0 com HCl 1M. </li><li> Preencha a solução com água até que o volume é 25ml. </li><li> Filtrar a solução usando papel Whatman. </li></ol></li></ol> 2. Dissecação das larvas Nota antes de começar: redes de microtúbulos, e em particular os dendritos sensorial, vai colapso rapidamente após o início da dissecção. CAhieving dissecção rápida em menos de cinco minutos, seguida pela fixação imediata são fatores chave para o sucesso deste protocolo. <ol><li> Lavar a larva na água e rapidamente movê-los para essa queda usando um laço de cabelo. </li><li> Orientar a larva até lado dorsal e no lado ventral no vidro. Nota: Esta orientação é examinar os neurônios aglomerado ventral. Para examinar os neurônios aglomerado dorsal, inverter a orientação. </li><li> Utilizar o centro de insetos pino a pino a extremidade anterior perto da boca ganchos. Coloque o pino perto do fim para obter melhores resultados. </li><li> Coloque uma gota de solução salina HL3.1 na câmara de dissecção. </li><li> Cortar a ponta posterior da larva fora com microtesoura. Nota: Este passo abre uma abertura na parte posterior da larva que irá permitir o acesso para a microtesoura (passo 2.7). </li><li> Pegar com a pinça a região do intestino que agora está saindo da abertura na extremidade posterior da larva. Retire cuidadosamente o intestino todo. </li><li> Coloque oponta de uma lâmina do microtesoura através da abertura e corte ao longo da linha mediana ventral para o anterior. </li><li> Utilizando os pinos de canto, pinos nos cantos agora livre, posterior primeiro, depois anterior. Simultaneamente esticar suavemente abrir o filé larval. </li></ol> 3. Fixação, o bloqueio, coloração e montagem de filetes larval Notas antes de começar: Todos os passos de fixação e coloração são realizadas na câmara de dissecção. Durante este processo, tome cuidado para não bater os pinos de insetos segurando a larva, pois isso pode levar a danos nos tecidos. Para evitar a experiência de secar, faça todos os passos coloração em um recipiente Tupperware pequena, rodeada por lenços umedecidos. <ol><li> Aspirar o Ca + + livre HL3.1 buffer usando uma ponta amarela. Imediatamente adicione o fixador usando outro pipetteman. </li><li> Pipeta suavemente para cima e para baixo para misturar o fixador com os traços remanescentes de Ca + + livre HL3.1 tampãona câmara. Imediatamente aspirado-lo e em seguida, adicione fixador fresco para dentro da câmara. </li><li> Incubar à temperatura ambiente por 20 minutos. </li><li> Lavar 10mins 6x no PBST (0,1% Triton X-100 em PBS). </li><li> Bloco com soro de cabra 5% em PBST por 20 minutos em temperatura ambiente. </li><li> Substitua a solução de bloqueio com anticorpos primários diluídos em soro de cabra 5% em PBST. Os anticorpos primários utilizados são mouse anti-α-tubulina (DM1A) e de ratos anti-CD8 (5H10) ambos diluídos a 1 / 1000. Nota: O investigador pode desejar substituir o rato tubulina anti-α-(DM1A) com o mouse anti-Futsch (22C10) 20,21 diluído 1 / 1000, em algumas circunstâncias (ver discussão). </li><li> Incubar overnight (por pelo menos 16h) a 4 ° C. </li><li> Lavar 10mins 6x no PBST. </li><li> Adicionar solução de anticorpo secundário diluído em soro de cabra 5% em PBST. Os anticorpos secundários utilizados são Alexa Fluor 488 cabra anti-camundongo IgG e Cy3 burro anti-rato IgG. Manter a amostra coberta para evitar fluoróforofoto-branqueamento 22. </li><li> Incubar quer no RT por duas horas, ou durante a noite a 4 ° C seguido por uma hora em temperatura ambiente. </li><li> Lavar 10mins 6x no PBST. </li><li> Coloque o filé larval no slide cutícula voltada para baixo, montado em glicerol 80%, e selar os lados da lamela com unha polonês para uma montagem de &#39;quick&#39;. Nota: Para limpar o tecido melhorou, e uma amostra permanente e estável, montagem em DPX como descrito anteriormente 23. </li></ol> 4. Resultados representativos: Fluorescente de coloração foi analisada sob um microscópio confocal. Nas Figuras 1-2, diferentes ramos dentro de um mandril dendrite tem organização do citoesqueleto diferentes. A Figura 1 mostra uma região do mandril de uma classe IV DA neurônio no estádio 1 º estágio larval. O mandril todo é marcada com mCD8:: KO e detectado com um anticorpo anti-CD8 e fluorescentes secundário (Cy3). Tubulina é detectada através de anticorpos anti α-tubulina e fluorescent secundário (Alexa Fluor 488). Os ramos principais são positivos para tubulina, alguns ramos laterais finas são tubulina-negativos. Um filme é um conjunto de cortes seriados através de uma coloração semelhante de uma classe I DA neurônio. A Figura 2 mostra uma região do mandril de uma classe IV DA neurônio corados com anticorpos contra Futsch e CD8 no estádio 1 º estágio larval. Os ramos principais são Futsch-positivos, alguns ramos laterais finas são Futsch-negativos. Figura 3. Traquéias na parede do corpo larval mostram uma organização de microtúbulos complexo. Filmes 2 e 3 mostram cortes seriados de coloração através dos músculos da parede do corpo e da traquéia. <img alt="Figura 1" src="/files/ftp_upload/3662/3662fig1.jpg" /> Figura 1. Fig. 1 mostra uma região do mandril de uma classe IV DA neurônio no estádio 1 º estágio larval. O mandril todo é marcada com mCD8:: KO e detectados utilizando anticorpos anti-CD8 e fluorescentes secundário (Cy3). Tubulina é detectada através de anti-&amp;-túbulosn anticorpo fluorescente e secundário (Alexa 488). AC painéis seqüenciais confocal z-seções (0.5μm),&#39;c-C &quot;, manchando de anticorpos único a partir do painel Exemplo de ramos com C. (amarelo ponta de seta) ou sem (roxo seta) microtúbulos são realçados. Red arrowheads microtúbulos destaque no subjacentes células epiteliais. <img alt="Figura 2" src="/files/ftp_upload/3662/3662fig2.jpg" /> Figura 2. Fig. 2 mostra uma região similar do mandril de um neurônio da classe IV corados com anticorpos contra Futsch e CD8 no estádio 1 º estágio larval. O mandril todo é marcada com mCD8:: KO e detectados utilizando anticorpos anti-CD8 e fluorescentes secundário (Cy3). Futsch é detectada através de anticorpos anti-Futsch e secundário fluorescente (Alexa 488). Os ramos principais são Futsch-positivos, alguns ramos laterais finas são Futsch-negativos. Exemplo de ramos com (seta amarela) ou sem (roxo seta) Futsch são realçados. Figura 3. Traquéia coradas utilizando o protocolo anti-tubulina descrito acima. Esta larva é de terceiro instar e dissecados como descrito anteriormente 23,24. Movie 3 mostra alargada cortes seriados do campo marcado por um quadrado. Movie 1. Seções Serial rastreamento coloração tubulina em todo o arbor dendríticas de uma classe que eu neurônio. O mandril todo é marcada com mCD8:: KO (Magenta) e detectados utilizando anticorpos anti-CD8 e fluorescentes secundário (Cy3). Tubulina (Green) é detectado com um anticorpo anti-α-tubulina e fluorescentes secundário (Alexa Fluor 488). Escala: de um lado da imagem de vídeo corresponde a 46.88μm na seção. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie1.avi">Clique aqui para ver o filme.</a> Filme 2. Seções Serial rastreamento coloração tubulina em bodmúsculos y parede de uma larva de terceiro instar. Tubulina é detectada através de anti-α-tubulina anticorpo fluorescente e secundário (Alexa Fluor 488). Escala: de um lado da imagem de vídeo corresponde a 46.88μm na seção. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie2.avi">Clique aqui para ver o filme.</a> Movie 3. Seções Serial rastreamento coloração tubulina em um corpo de parede da traquéia de uma larva de terceiro instar, marcado com um quadrado no Figure.3. Tubulina é detectada através de anti-α-tubulina anticorpo fluorescente e secundário (Alexa Fluor 488). Escala: de um lado da imagem de vídeo corresponde a 46.88μm na seção. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie3.avi">Clique aqui para ver o filme.</a>

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Para entender o quão complexo formas de células são alcançados, é importante ser capaz de precisão organização de microtúbulos ensaio. Aqui nós descrevemos um método etiquetagem robusta imuno ensaio para a organização de microtúbulos dos dendritos arborização dendrítica de neurônios sensoriais. Além da coloração neurônios sensoriais, este método obtém coloração imuno robusto de traquéia, músculos e tecidos outra parede do corpo. Nós usamos esse protocolo para exa...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Catálogo número </th><th> Comentários (Opcional) </th></tr><tr><td> Fórceps </td><td> Dumont </td><td> 11251-20 </td><td></td></tr><tr><td> Microtesoura </td><td> FST </td><td> 15000-08 </td><td></td></tr><tr><td> Do mouse anti-α-tubulina (Clone: ​​DM1A) </td><td> Sigma </td><td> T9026 </td><td> Diluição 1 / 1000 </td></tr><tr><td> Rato anti-Futsch (Clone: ​​22C10), sobrenadante </td><td> Desenvolvimento Estudos Hibridoma Banco </td><td> 22C10 </td><td> Diluição 1 / 1000 </td></tr><tr><td> Rat anti-CD8 (Clone: ​​5H10) </td><td> Caltag </td><td> MCD0800 </td><td> Diluição 1 / 1000 </td></tr><tr><td> Alexa Fluor 488 anti-mouse IgG </td><td> Invitrogen </td><td> A-11001 </td><td> Diluição 1 / 500 </td></tr><tr><td> Cy3 IgG anti-rato </td><td> Immunoresearch Jackson </td><td> 712-166-150 </td><td> Diluição 1 / 200 </td></tr></tbody></table>

immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the drosophila larva body wall

Para entender como as diferenças de formas complexas de células são alcançados, é importante seguir precisamente organização de microtúbulos. A Drosophila parede do corpo larval contém vários tipos de células que são modelos para o estudo celular e morfogênese do tecido. Por exemplo traquéias são usados ​​para examinar morfogênese tubo 1, ea arborização dendrítica (DA) neurônios sensoriais da larva Drosophila tornaram-se um sistema primário para a elucidação da geral e neurônio-específico de classe mecanismos de diferenciação 05/02 dendríticas e degeneração 6 . A forma de ramos dendríticos podem variar significativamente entre as classes de neurônios, e até mesmo entre os diferentes ramos de um único neurônio 7,8. Estudos genéticos em neurônios DA sugerem que a organização do citoesqueleto diferencial pode subjacentes diferenças morfológicas na forma ramo dendríticas 4,9-11. Nós fornecemos um método de etiquetagem robusta imunológica a umssay in vivo organização de microtúbulos em DA neurônio sensorial dendrite arbor (Figuras 1, 2, Movie 1). Este protocolo ilustra a dissecção e imunomarcação de larva de primeiro ínstar, uma fase em conseqüência dendrito ativo neurônio sensorial e organização ramificação está ocorrendo 12,13. Além da coloração neurônios sensoriais, este método obtém etiquetagem robusta de organização de microtúbulos nos músculos (Filmes 2, 3), traquéia (Figura 3, Movie 3), e tecidos outra parede do corpo. É valioso para pesquisadores que desejam analisar a organização de microtúbulos in situ na parede do corpo quando se investiga os mecanismos que o tecido de controle e forma da célula.

Watch this Scientific Journal Video about Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall at JoVE.com

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

Para compreender como as formas de células complexas, como dendritos neuronais, são alcançados durante o desenvolvimento, é importante ser capaz de precisão organização de microtúbulos ensaio. Aqui nós descrevemos um método imuno etiquetagem robusta para analisar a organização dos microtúbulos dos dendritos arborização dendrítica neurônio sensorial, traquéia, músculos e outros<em&gt; Drosophila</em&gt; Larva tecidos parede do corpo.

Labeling imuno dos microtúbulos no Dendrites neurônio sensorial, traquéias, e Músculos no Drosophila Wall Corpo Larva

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

immunohistological-labeling-microtubules-sensory-neuron-dendrites

Research

JoVE Journal

Neuroscience

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

12.3K visualizações.  RIKEN Brain Science Institute. Para compreender como as formas de células complexas, como dendritos neuronais, são alcançados durante o desenvolvimento, é importante ser capaz de precisão organização de microtúbulos ensaio. Aqui nós descrevemos um método imuno etiquetagem robusta para analisar a organização dos microtúbulos dos dendritos arborização dendrítica neurônio sensorial, traquéia, músculos e outros Drosophila Larva tecidos parede do corpo.

Vídeo: Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

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

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

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

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

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

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

Neurociência

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 ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Labeling fluorescentes retrógrada Permite alvejado Extracelular Gravação Single-unidade dos neurônios identificados<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Marcação de células individuais no Sistema Nervoso Central de<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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 ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

Comportamentos olfativos ensaiadas pelo monitoramento do computador de<em&gt; Drosophila</em&gt; Em um olfatômetro de quatro quadrantes

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Rotulagem Balística de Neurônios Piramidais em Fatias Cerebrais e na Cultura Celular Primária

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Determinação semi-quantitativa da densidade do neurônio dopaminérgico na Substantia Nigra dos Modelos de Roedores usando análise automatizada de imagem

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

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

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

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

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

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