SG é apoiado por fundos da Universidade Estadual de Nova York em Buffalo e de John R. Oishei Foundation.

<html> 1. Preparação dos Reagentes <ol><li> Prepare o Buffer PEMFA usando Pipes 100mM, 2mM EGTA, e 1 mM MgSO 4. Ajustar o pH do tampão a 7,0. O buffer PEMFA é esterilizado por filtração e armazenado a 4 ° C. </li><li> Prepare PBT, adicionando 1 ml de Triton X-100 a 100ml 1x PBS (Phosphate Buffered Saline) (pH 7,0). Armazenar a 4 ° C. </li><li> Prepare BSA 5% (albumina bovina) com NaAcide 0,01% em 1x PBS (pH 7,0). </li><li> Prepare a solução salina usando 0,7% de NaCl e 0,03% Triton X-100 em água deionizada. </li><li> Prepare o fixador, combinando quatro partes tampão PEMFA com 1 parte de formaldeído 37%. </li></ol> 2. Coleta de embriões <ol><li> Prepare uma gaiola para a cepa Drosophila de interesse, colocando&gt; 100 moscas em uma garrafa de plástico contendo um adaptador de alimentação com placa de agar. </li><li> Deixe a gaiola de moscas no escuro à temperatura ambiente por 72 horas, mudando as placas a cada 8-12 horas, de modo que as moscas se acostumar à gaiola e não mantenha seus ovos. </li><li> Para o experimento coletar embriões para 12 horas. Remova a placa de Petri do recipiente e substituí-lo. Pegue a placa de Petri removido e lavar os embriões fora do prato utilizando a solução salina e um pincel limpo. </li><li> Coletar os embriões em uma peneira fina. Enxaguar com soro fisiológico para remover qualquer excesso de levedura. </li><li> Usando uma espátula delicadamente pegar os embriões e colocá-las num frasco de vidro pequeno com tampa. Adicionar cerca de 1ml de solução salina. </li></ol> 3. Remoção das membranas Chorion e Vitelline e Fixação <ol><li> Após a coleta dos embriões, preparar a solução de heptano-Fix, combinando 50% heptano e fixador de 50%. A solução será em camadas com heptano na parte superior eo fixador na parte inferior. </li><li> No frasco de vidro de lavagem dos embriões, incubando-as na solução salina por 5 minutos. Remover salina em excesso com uma pipeta Pasteur. </li><li> Enxágüe os embriões com água deionizada. </li><li> Dechorionate os embriões, incubando-as em água sanitária 50% por 3 minutos. </li><li> Enxágüe os embriões cuidadosamente com água deionizada. </li><li> Fixar os embriões, incubando-as por 30 minutos na solução de heptano-Fix, agitando suavemente. </li><li> Com uma pipeta Pasteur substituir o (camada inferior) fixador com metanol deixando o heptano no tubo. Agite vigorosamente para remover a membrana vitelina. Os embriões devitellinized vai afundar até o fundo do tubo. </li><li> Coletar os embriões em um frasco de vidro novo, utilizando uma pipeta Pasteur. Enxágüe os embriões cinco vezes com metanol, cada vez incubar os embriões por 5 minutos. </li></ol> 4. Coloração de anticorpos, montagem, e Visualização <ol><li> Hidratar os embriões em uma mistura de 50% de solução PBT e metanol 50% por 5 minutos. Em seguida, hidratar em 100% PBT solução por 5 minutos. </li><li> Bloco os embriões, incubando-as em BSA 5% com a solução NaAcide 0,01% para 1 hora a 4 ° C. </li><li> Embriões estão agora prontos para a coloração de anticorpos. Prepare o anticorpo primário, diluindo-lo para a concentração adequada usando BSA 5% com a solução NaAcide 0,01%. Nós usamos um anticorpo contra a proteína cadeia cisteína (CSP), uma proteína vesicular sináptica e um anti-anticorpo HRP ou cavalo peroxidase de rabanete, que reconhece uma porção específica de carboidratos neuronal presente no glicoproteínas de superfície e etiquetas todos os neurônios. </li><li> Incubar os embriões durante a noite a 4 ° C na solução de anticorpo primário. </li><li> No dia seguinte, lave os embriões 10 vezes com PBT ao longo de uma hora. </li><li> Preparar a solução de anticorpo secundário diluindo a concentração adequada. Usamos anticorpos Alexa secundário na concentração de 1:200. </li><li> Incubar os embriões durante duas horas no 4 ° C na solução de anticorpo secundário. </li><li> Lavar os embriões 10 vezes com PBT ao longo de uma hora. </li><li> Incubar os embriões durante a noite em Vectashield Meio de Montagem. </li><li> Monte os embriões em uma lâmina de vidro e selar a lamínula com unha polonês. Visualize embriões utilizando um microscópio de fluorescência. </li></ol> 5. Resultados representante <img src="/files/ftp_upload/2150/2150fig1a.jpg" alt="Figura 1" /> CSP <img src="/files/ftp_upload/2150/2150fig1b.jpg" alt="Figura 1" /> HRP-FITC <img src="/files/ftp_upload/2150/2150fig1c.jpg" alt="Figura 1" /> Fundiram Figura 1: A Representante imagens mostrando o SNC na fase embrionária 16-17, utilizando anticorpos contra o CSP (vermelho) e HRP (verde). Observe a coloração distinta da comissura. <img src="/files/ftp_upload/2150/2150fig2a.jpg" alt="Figura 2" /> CSP <img src="/files/ftp_upload/2150/2150fig2b.jpg" alt="Figura 2" /> HRP-FITC &quot;/&gt; Fundiram Figura 2: Um representante de imagens mostrando o PNS embrionárias na fase 16-17 usando anticorpos contra o CSP (vermelho) e HRP (verde). Observe a padrões repetidos de neurônios periféricos.

<ol>
	<li>Bodmer, R., Barbel, S., Sheperd, S., Jack, J. W., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+sensory+organs+by+mutations+of+the+cut+locus+of+D.+melanogaster.">Transformation of sensory organs by mutations of the cut locus of D. melanogaster.</a> Cell. 51, 293-307 (1987).</li><li>Bodmer, R., Carretto, R., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+of+the+peripheral+nervous+system+in+Drosophila+embryos:+DNA+replication+patterns+and+cell+lineages.">Neurogenesis of the peripheral nervous system in Drosophila embryos: DNA replication patterns and cell lineages.</a> Neuron. 3, 21-32 (1989).</li><li>Campos-Ortega, J. A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Embryonic Development of Drosophila melanogaster. , (1985).</li><li>Carroll, S. B., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+the+fushi+tarazu+protein+during+Drosophila+embryogenesis.">Localization of the fushi tarazu protein during Drosophila embryogenesis.</a> Cell. 43, 47-57 (1985).</li><li>Dambly-Chaudi&#232;re, C., Ghysen, A., Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+connections+between+imaginal+discs+in+Drosophila.">Muscle connections between imaginal discs in Drosophila.</a> Developmental Biology. 113, 288-294 (1986).</li></ol>

Não há conflitos de interesse declarados.

O embrião Drosophila é um sistema poderoso para investigar as mudanças celulares e moleculares durante o desenvolvimento neuronal. Neste protocolo temos demonstrado como preparar embriões montar toda a imuno-histoquímica. Nós adaptamos este protocolo a partir do protocolo descrito no Carroll e Scott, 1985. Este protocolo também pode ser adaptado para testar outros anticorpos neuronal, mas a otimização de anticorpos deve ser feito. Além disso, defeitos de desenvolvimento do PNS embrionárias e sistema ...

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<td>37% formaldehyde</td>
<td>&nbsp;</td>
<td>Fisher</td>
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<td>Developmental Studies Hybridoma Bank</td>
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<td>HRP-FITC</td>
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<td>Jackson ImmunoResearch Laboratories</td>
<td>323-095-021</td>
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<td>Alexa Fluor 568 Goat Anti-Mouse IgG</td>
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<td>Invitrogen</td>
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visualization of the embryonic nervous system in whole-mount drosophila embryos

O embrião Drosophila é um sistema modelo atraente para investigar as bases celulares e moleculares do desenvolvimento neuronal. Aqui nós descrevemos o procedimento para a visualização de Drosophila sistema nervoso embrionário utilizando anticorpos para proteínas neuronal. Uma vez que todo o embrião nervoso periférico e sistema nervoso central estão bem caracterizados no nível de células individuais (Dambly-Chaudière et al, 1986;.. Bodmer et al, 1987;. Bodmer et al, 1989), qualquer aberrações para estes sistemas podem ser facilmente identificados através de anticorpos para diferentes proteínas neuronal. Os embriões em desenvolvimento são coletados em determinados momentos para garantir que os embriões estão em fase de desenvolvimento adequado para visualização. Após a coleta, as camadas externas do embrião, a membrana cório eo envelope vitelínico que envolve o embrião, são removidos antes da fixação. Embriões são então incubados com anticorpos neuronal e visualizados usando fluorescente etiquetado anticorpos secundários. Embriões em estágios 12-17 são visualizados para acessar o sistema nervoso embrionário. No estágio 12 a banda germe CNS começa encurtamento e por etapa 15, o padrão definitivo da comissura foi alcançado. Por estágio 17 os contratos do SNC e do SNP está totalmente desenvolvido (Campos-Ortega et al. 1985). Assim, mudanças no padrão do PNS e sistema nervoso central pode ser facilmente observado durante estes estágios de desenvolvimento.

Watch this Scientific Journal Video about Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos at JoVE.com

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

Nós descrevemos o procedimento para preparar encenado<em&gt; Drosophila</em&gt; Embriões para a visualização do sistema nervoso embrionário durante a embriogênese.

Visualização do Sistema Nervoso embrionárias no Whole-mount Drosophila Os embriões

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

visualization-embryonic-nervous-system-whole-mount-drosophila

Research

JoVE Journal

Neuroscience

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

13.2K visualizações.  SUNY-University at Buffalo. Nós descrevemos o procedimento para preparar encenado Drosophila Embriões para a visualização do sistema nervoso embrionário durante a embriogênese.

Vídeo: Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

Preparação Monte todo o Adulto Drosophila Cordão nervoso ventral para injeção gigante de fibra Dye

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Neurociência

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 ...

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 ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

Um Paradigma para Investigar Lesão de reparação do sistema nervoso central em<em&gt; Drosophila</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Entrega intraventricular direta de medicamentos para o Sistema Nervoso Central Rodent

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

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 ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo Optogenetic estimulação do sistema nervoso central de roedores

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

Monte todo Rotulagem de Cilia na Olfativo Sistema de Ratos Principal

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca 2+ Imagem do Sistema Nervoso Entérico

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Um de uma etapa simples protocolo de dissecação para Whole mount-Preparação do Adulto<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Visualização do Sistema Nervoso embrionárias no Whole-mount Drosophila Os embriões

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