Gostaríamos de agradecer a todos os membros de nosso laboratório para os seus conselhos, discussões e comentários úteis sobre o manuscrito. Gostaríamos também de agradecer particularmente Marta Koch e Bassem Hassan para a excelente sugestão que esperar até 1,5 horas APF antes de dissecar o cérebro para cultura em experimentos de remodelação. Agradecemos também a facilidade de imagem NHGRI para o uso de seu microscópio confocal. Este trabalho foi financiado pelo Programa de Neurociência Básica do Programa de Pesquisa Intramural NINDS, NIH (Z01 NS003106).

A. Preparação de Mídia <ol><li> Preparar meio completo pelo filtro esterilizante-Drosophila Schneider meio suplementado com 10% de soro fetal bovino e 1% de penicilina / estreptomicina. </li><li> Os meios devem ser preparados de fresco de cada vez. Alternativamente, uma nova porção da pré-congelados meios de comunicação podem ser descongelado antes de cada utilização. </li><li> Antes da cultura, adicionam-se 10 ug / ml de insulina e 2 ecdisona ug / ml para os meios de comunicação. As drogas foram preparadas como concentrados e stocks congelados em pequenas alíquotas. Alíquotas não foram descongeladas recongelados. </li><li> Todas as soluções de trabalho deve ser mantida a 25 ° C. </li></ol> A cultura de Brains larval 1. Seleção e Dissecção da Brains larval <ol><li> Adicionar 500 ul de mídia para um Millicell 12 mm inserção de cultura de célula. Os cérebros dissecados será colocado no estas inserções para facilitar imunocoloração fácil. </li><li> Adicionar uma pequena quantidade de meios de preparados para um vidro de relógio dissecção. </li><li> Ucantar uma pequena espátula, pegar vagando larvas de terceiro estádio e colocá-los no vidro de relógio. (Supomos que o método de cultura ex vivo se aplica igualmente bem a primeira ou segunda larvas, mas não testei isso.) </li><li> Segurando a extremidade posterior da larva com um par de pinças, cuidadosamente abrir a extremidade anterior com um segundo par de # 5 fórceps. </li><li> Rapidamente dissecar o cérebro, mantendo o cordão nervoso ventral intacto. </li></ol> 2. Cultura ex vivo de Dissecada Brains larval <ol><li> Utilizando pontas de pré-molhadas pipeta, transferir o cérebro cuidadosamente nas inserções de cultura de células pré-preenchidos com meios de comunicação. </li><li> Cérebros de transferência, em media, para uma incubadora a 25 ° C até que o desejado tempo de ponto é atingido. Este método funciona de forma eficiente para até 48 h em cultura. </li><li> Substitua a mídia menos 8 horas. Além disso, substituir metade dos meios de comunicação a cada 5-6 horas. </li></ol> 3. Manipulação farmacológica doBrains larval Este método de cultura ex vivo pode ser usada para farmacologicamente corroboram interacções genéticas previamente demonstrada no nosso laboratório 1. <ol><li> Adicionar 100 roscovitina mM e 100 mM olomoucina a mídia preparados. </li><li> Transferência cérebros dissecados para essa mídia. </li><li> Cérebros de cultura a 25 ° C durante 24 horas. </li><li> Substitua a mídia menos 8 horas. Além disso, substituir metade dos meios de comunicação (com drogas) a cada 5-6 horas. </li></ol> A cultura de Brains pupais 1. Seleção de pupas para Dissecção <ol><li> Marcar pupas branco em garrafas ou frascos. </li><li> Só escolher pupas, que são completamente branco. Pupas com qualquer tipo de coloração não deve ser incluída. </li><li> Esta fase é de 0 horas após a formação pupário (APF). </li><li> Estes pupas estará pronto para dissecção 1,5 horas após a marcação. Esta etapa é crucial para garantir que a poda de desenvolvimento ocorre em ex vivo </em&gt; cultura. </li></ol> 2. Dissecção de pupas <ol><li> Adicionar 500 ul de mídia para um Millicell 12 mm inserção de cultura de célula. (Os cérebros dissecados será colocado no estas inserções para facilitar imunocoloração fácil). </li><li> Uma vez que o desejado tempo de ponto-tiver sido atingido, adicionar uma pequena quantidade de meios de preparados para um vidro de relógio dissecção. </li><li> Usando uma espátula pequena, molhada, pegar pupas anteriormente marcado para dissecção e colocá-los no vidro de relógio. </li><li> Segurando a extremidade posterior do pupas com um par de pinças, cuidadosamente abrir a extremidade anterior do processo pupa com um segundo par de # 5 fórceps. </li><li> Rapidamente dissecar o cérebro, mantendo o cordão nervoso ventral intacto e em anexo. </li></ol> 3. A cultura ex vivo de Dissecada Brains pupais <ol><li> Transferir o cérebro cuidadosamente nas inserções de cultura de células pré-preenchidos com meios de comunicação. </li><li> Cérebros de transferência, em media, para uma incubadora a 25 ° C até o desejado tempo de ponto é atingido. Utilizou-se este método para até 10 horas em cultura para a experiência mostrado, mas pode ser estendida para períodos mais longos de observação e tratamento, se necessário. </li><li> Para prazos mais de 8 pontos APF horas, substituir a mídia a cada 5-6 horas. </li></ol> A partir deste passo em diante, o protocolo permanece inalterado, tanto para larvas e pupas. B. Fixação Brains Dissecada <ol><li> Uma vez que o desejado tempo de ponto é atingido, remover meios de comunicação e adicionar 500 uL de formaldeído a 4% em PBS 1x com 0,5% de Triton X-100 (PBST) durante 30 minutos, substituindo com formaldeído fresca depois de 15 minutos. </li><li> Certifique-se de não permitir que cérebros para secar ao substituir a mídia com fixador. </li><li> Lavar formaldeído com quatro lavagens de 10 minutos de 1x PBS com 0,5% tritonX-100 (PBST). </li></ol> C. Coloração Brains fixos <ol><li> Depois de formaldeído foi lavada, bloquear cérebros durante 1 hora numa solução de bloqueio de 1x PBS com 1% NormalSoro de cabra e 1% albumina sérica bovina. </li><li> Adicionar anticorpos primários formados em solução de bloqueio. </li><li> Cérebros incubar durante a noite a 4 ° C. </li><li> Na manhã seguinte, lave anticorpos primários com quatro lavagens 10 minutos de PBST. </li><li> Adicionar anticorpos secundários formados em solução de bloqueio. </li><li> Deixar à temperatura ambiente durante 4 horas. </li><li> Lavar anticorpos secundários com quatro lavagens 10 minutos de PBST. </li></ol> D. Montagem Brains fixos e corado para microscopia <ol><li> Equilibrar cérebros na mídia Vectashield montagem para 1 hora. </li><li> Monte cérebros em lâminas de vidro, utilizando os meios de comunicação Vectashield de montagem. </li><li> Colocar fichas de vidro em ambos os lados dos cérebros antes da montagem do deslizamento da tampa. Isso impede que os cérebros de ser esmagado, mantendo amostra fina o suficiente para digitalizar todos os planos focais com um microscópio confocal. Cérebros eram aproximadamente orientados sobre a montagem para facilitar a visualização das características de interesse. Quando necessário,imagens do cérebro foram rodados com software Imaris para proporcionar uma visão ideal para documentação e medição. </li><li> Vedação da tampa desliza com unhas polonês claro. </li><li> Loja desliza longe da luz. </li></ol> Resultados E. Representante A Figura 1 mostra um painel de terceiro instar-cérebros de larvas de (A) do tipo selvagem moscas e (B) moscas que expressam um derivado dominante negativo de CDK5 (Cdk5DN) (Connell-Crowley et ai., 2000). Ambos expressam actina-GFP (verde) nos neurônios gama MB. Tal como descrito anteriormente (Trunova et al., 2011), há uma região em que os axônios proximais dos neurónios do tipo selvagem gama que falha a acumular-se actina-GFP, mostrando assim como um &quot;zona clara&quot; (A, o suporte) e, fasciclina proteína 2 (vermelha) se acumula nos axônios apenas até a borda ventral desta zona (linha horizontal branca). A linha pontilhada no terceiro painel da Figura 1 (A) indica a posição do MB, realçando o actona &quot;zona clara&quot; e sua borda ventral. Em moscas que expressam Cdk5DN, a zona de actina claro está ausente (B) e fasciclina 2 (FasII) imunorreactividade espalha dorsalmente por aquela região. (Note que os números variáveis ​​de FasII positivo projeto axônios horizontalmente em toda a MB, estes não são relevantes para o fenótipo). Barra de escala representa 20 microns. Utilizando este método de cultura ex vivo, foram tratados de tipo selvagem moscas com uma combinação de roscovitina e olomoucina, inibidores farmacológicos de CDK5 actividade. A Figura 2 mostra um painel de terceiro instar-cérebros de larvas de controlo (A) e tratados com droga (B ) do tipo selvagem moscas. Semelhante ao moscas que expressam um dominante negativo CDK5 construto, os cérebros tratados com inibidores de CDK5 durante 24 horas, mostram a perda característica de actina zona clara e mudança na fronteira FasII (parênteses rectos). Linhas brancas mostram a posição de tipo selvagem fronteira FasII. Barra de escala representa 20 microns. FIGUre 3 mostra uma série representativo de organismos de cogumelo de Drosophila de 0-10 horas APF, rotulado com membrana-alvo mCD8-GFP (verde). Parêntesis indicam o cálice, isto é, as projecções dendríticas do MB. Antes de metamorfose, pode-se ver axónios MB coursing dorsoventralmente em feixes de espessura, bem como uma &#39;neblina&#39; de sinal fluorescente resultante da densa dendrítica mandril (suporte). O painel A mostra cérebros dissecados à hora indicada. Note-se a poda de dendritos na região indicada pelos parênteses, de tal forma que por 6-8 hrs APF, a neblina de sinal dendrítica se foi (embora o sinal axonal não é afetada). Brains no painel B foram dissecados em 0 horas APF, antes do interno ecdisona pico (Truman e Riddiford, 2002), e ex vivo de cultura, como indicado. Estes mostram nenhuma poda de desenvolvimento de dendritos, mas sim o sinal dendrítica persiste em 10 horas da APF. Para contornar isso, pupas são marcados em 0 APF horas, mas dissecado em 1,5 horas APF e culta. Painell C mostra uma série representativo destes corpos de cogumelo de Drosophila a partir de 0-10 APF horas. Tal como nas amostras não cultivadas, o sinal dendrítica é indetectável por 8 hr APF. Barra de escala representa 20 microns.

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<html> Crónicas manipulações genéticas da estrutura e função dos tecidos, incluindo mutações e expressão de transgenes, produzem tipicamente uma combinação de efeitos imediatos e tardios consequências indirectas que podem ser muito difíceis de separar. Complementando métodos genéticos com a farmacologia pode facilitar o interrogatório do curso de tempo atrás de um fenótipo. Por exemplo, esta tem sido uma poderosa abordagem para dissecar as contribuições dos diferentes componentes do citoesqueleto dura...

<table border="1"><tbody><tr><td> Nome do Reagente </td><td> Companhia </td><td> Número de Catálogo </td><td> Concentração Final </td></tr><tr><td> Drosophila Schneider Mídia </td><td> Invitrogen </td><td> 21720-024 </td><td></td></tr><tr><td> Soro Fetal Bovino (inactivado pelo calor) </td><td> Invitrogen </td><td> 10082-147 </td><td> 10% </td></tr><tr><td> Penicilina / estreptomicina </td><td> Invitrogen </td><td> 15140-122 </td><td> 1% </td></tr><tr><td> Insulina </td><td> Invitrogen </td><td> 12585-014 </td><td> 10μg/ml </td></tr><tr><td> Ecdisona </td><td> Sigma-Aldrich </td><td> H5142 </td><td> 2μg/ml </td></tr><tr><td> Roscovitina </td><td> Sigma-Aldrich </td><td> R7772 </td><td> 100 um </td></tr><tr><td> Olomoucina </td><td> Sigma-Aldrich </td><td> O0886 </td><td> 100 um </td></tr><tr><td> Soro de cabra normal </td><td> Invitrogen </td><td> 50062Z </td><td> 1% </td></tr><tr><td> Albumina de Soro Bovino </td><td> Fisher Scientific </td><td> M9501 </td><td> 1% </td></tr><tr><td> Formaldeído </td><td> Sigma-Aldrich </td><td> D4551 </td><td> 4% </td></tr><tr><td> Triton-X 100 </td><td> Fisher Scientific </td><td> BP151 </td><td> 0,5% </td></tr><tr><td> Tampão fosfato salino </td><td> Invitrogen </td><td> 70011-044 </td><td> 1X </td></tr><tr><td> Vectashield </td><td> Vector Laboratories </td><td> H1000 </td&gt; <td></td></tr><tr><td> Cultura Millicell Insere celular </td><td> Millipore </td><td> PI8P01250 </td><td></td></tr><tr><td> Multidish, 4 bem </td><td> Thermo Scientific </td><td> 176740 </td><td></td></tr><tr><td> Dumont # 5 Pinça </td><td> Belas Ferramentas Ciência </td><td> 11251-20 </td><td></td></tr><tr><td> Lâminas de Microscopia </td><td> Fisher Scientific </td><td> 12-544-7 </td><td></td></tr><tr><td> Capa de vidro </td><td> Fisher Scientific </td><td> 12-548-5M </td><td></td></tr></tbody></table>

ex vivo culturing of whole, developing drosophila brains

Nós descrevemos um método para a cultura ex vivo de cérebros inteiros de Drosophila. Isto pode ser utilizado como um contraponto crónicas manipulações genéticas para investigar a biologia celular e desenvolvimento de estruturas cerebrais centrais, permitindo agudas intervenções farmacológicas e de imagiologia vivo de processos celulares. Como um exemplo da técnica, antes trabalhar a partir de nosso laboratório 1 demonstrou que um compartimento anteriormente não reconhecida subcelular situa-se entre os compartimentos axonais e somatodendritic de axónios do cérebro central Drosophila. O desenvolvimento deste compartimento, referido como o segmento de axónio inicial (AIS) 2, foi mostrado geneticamente depender da quinase neurónio específico dependente de ciclina, CDK5. Nós mostramos aqui que o tratamento ex vivo de cérebros de tipo selvagem de Drosophila larvais com a CDK5-farmacológico específico inibidores roscovitina e olomoucina 3 provoca alterações agudas em organização da actina, ena localização da proteína da superfície celular fasciclina 2, que imitam as alterações observadas nos mutantes que não possuem actividade CDK5 geneticamente. Um segundo exemplo da técnica de cultura ex vivo está prevista a remodelação das ligações do corpo de cogumelo embrionário (MB) neurônios gama durante a metamorfose de larva a adulto. O corpo de cogumelo é o centro da aprendizagem e memória olfativa na mosca 4, e esses neurônios gama podar os galhos axonais e dendríticas durante o desenvolvimento pupal e então re-ramos se estendem em um ponto no tempo mais tarde para estabelecer o padrão de inervação adulto 5. Poda destes neurónios do MB tem sido demonstrado que ocorrem através degeneração local de ramos neurites 6, por um mecanismo que é desencadeada por ecdisona, uma hormona esteróide, actuando no receptor de ecdisona B1 7, e que é dependente da actividade do ubiquitina-proteassoma sistema 6. O nosso método de cultura ex vivo pode be usado para interrogar ainda mais o mecanismo de remodelação de desenvolvimento. Descobrimos que na configuração de ex vivo cultura, os neurónios gama do MB recapitulou o processo de poda do desenvolvimento com um curso de tempo semelhante ao que in vivo. Era essencial, no entanto, de esperar até 1,5 horas após a formação pupário antes explantar o tecido para que as células de cometer irreversivelmente a metamorfose; dissecação dos animais no início da pupariation levou a metamorfose pouca ou nenhuma em cultura. Assim, com a modificação apropriada, a abordagem de cultura ex vivo pode ser aplicado para estudar dinâmica bem como os aspectos de estado estacionário da biologia cérebro central.

Watch this Scientific Journal Video about Ex vivo Culturing of Whole, Developing Drosophila Brains at JoVE.com

Ex vivo Culturing of Whole, Developing Drosophila Brains

Este artigo descreve um método pelo qual se pode imitar<em&gt; In vivo</em&gt; Desenvolvimento do<em&gt; Drosophila</em&gt; Corpo cogumelo em uma<em&gt; Ex vivo</em&gt; Sistema de cultura.

A cultura Ex vivo de todo, o desenvolvimento cerebral de Drosophila

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

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JoVE Journal

Neuroscience

Ex vivo Culturing of Whole, Developing Drosophila Brains

13.0K visualizações.  National Institute of Neurological Disorders and Stroke. Este artigo descreve um método pelo qual se pode imitar In vivo Desenvolvimento do Drosophila Corpo cogumelo em uma Ex vivo Sistema de cultura.

Vídeo: Ex vivo Culturing of Whole, Developing Drosophila Brains

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

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

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

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

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 ...

Neurociência

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Em Eletroporação vivo de Retina do rato em desenvolvimento

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

Ex vivo imagens ao vivo de divisões celulares simples no mouse neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

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 ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultâneo<em&gt; Ex vivo</em&gt; Teste Funcional de Dois retinas por<em&gt; In vivo</em&gt; Sistema Eletrorretinograma

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Ex Vivo optogenética Dissection de Circuitos medo no cérebro Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

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 ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Ex Utero Eletroporação e culturas Organotypic fatia do cérebro embrionário Mouse para imagens ao vivo de migração gabaérgica interneurônios

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Imagem de cálcio para visualizar as respostas do cérebro a sinalização endócrina em Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

Análise metabólica do melanogaster da drosófila Larval e adulto cérebros

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...