Este trabalho foi financiado por uma bolsa de Ehrman para HY. H., Jane Coffin Childs Memorial Fund para a comunhão de Pesquisa Médica de pós-doutorado para RJJ, NIH Grant F32EY016309 para DV, bolsa de Nova York University Dean Dissertação de DJ, NIH GrantR01 EY13010 em CD e uma bolsa DFG a JR (RI 2208/1- 1). Agradecemos Nina Vogt e Pamela Boodram para comentários sobre o manuscrito.

1. Introdução Neste vídeo, descrevemos métodos para dissecção da retina e imunohistoquímica em três períodos de desenvolvimento definidos: o terceiro ínstar, o midpupal ea fase adulta. Embora nosso protocolo também funciona para outras fases de pupa (para mais detalhes sobre etapas anteriores, consulte 16), que escolheu o palco midpupal, pois é ideal para imagens de todos os PRs em um plano focal e seus núcleos são facilmente identificáveis, o que facilita a visualização de padrões de expressão do factor de transcrição. A dissecção da retina de pupa tardia é muito semelhante à dissecção de retinas adultas. Primeiro, vamos demonstrar como coleta de larvas e pupas em estágios desejados. Então, vamos explicar como obter dissecções ideais de larvas 17-18, midpupal e retinas adultas. Em segundo lugar, demonstrar como visualizar o desenvolvimento PR nestas fases usando microscopia confocal e imunohistoquímica. O período de desenvolvimento dos insetos é dependente da temperatura. Para facilitar o preparo reprodutível de larvas e pupas, recomendamos levantar todas as ações da mosca a 25 ° C sob uma hr/12 hr 12 ciclo de luz / escuridão. Terceiro-larvas: Cerca de 96 horas após a postura de ovos (a 25 ° C), final do terceiro instar alimentação parada larvas e vagar nas paredes do frasco de alimentos, onde eventualmente pupate. Use uma escova macia ou uma pinça para remover suavemente a vaguear larva do terceiro instar da parede do frasco de alimentos. Pupas de 50% (&#39;midpupae&#39;) são observados cerca de 48 horas após a fase de pupa. Para o tempo exato, você pode circular apenas formado pupas branco com um marcador no frasco de alimentos. Alternativamente, mova todos pupas branco para um novo frasco para obter um frasco de teste uniforme (para teste de pupas utilizando critérios morfológicos, consulte 19). Depois de 48 horas, cuidadosamente reaisove o midpupae do frasco usando uma pinça. Moscas adultas: anestesiar moscas adultas jovens (2-5 dias após a eclosão) sobre uma almofada de CO 2. Remover cabeças usando uma pinça e armazená-los em um copo bem gelado prato contendo 1x PBS. 3. Procedimento dissecção (cerca de 1-2 horas por Experiment) Após a coleta da amostra, coloque-os em uma gota de PBS gelado em um prato dissecção Sylgard. Dissecar cerca de 10-15 retinas por genótipo. Por favor, consulte as Fichas de Segurança, bem como Saúde Ambiental de sua instituição e Secretaria de Segurança para o correcto manuseamento dos reagentes e equipamentos (ver tabelas ao final deste protocolo). 1. Larval Discos Eye imaginárias <ol><li> Localizar a extremidade anterior da larva, que contém os ganchos de boca. Use uma pinça para pressionar a parte do meio da larva suavemente contra a base do prato Sylgard. Use another uma pinça para agarrar os ganchos na boca e puxe devagar e com cuidado para fora do corpo. Descarte a parte principal do corpo e usar os aparelhos bucais para segurar a parte anterior a fim de remover o tecido estranho a partir dos discos imaginais que permanecem em anexo para o cérebro. </li><li> Transferir cérebro com discos imaginais, agarrando os ganchos na boca com um fórceps para 1x PBS em um prato de vidro de três poços. Executar o passo de fixação (passo 4.1.). </li><li> Após a fixação, remover as glândulas salivares, tecido adiposo e puxe o cérebro para longe do disco imaginal olho-antenais. Para melhorar a acessibilidade dos &quot;difíceis&quot; anticorpos, a membrana peripodial, que está localizado sobre a superfície do epitélio, podem ser removidos, mantendo-se a parte da antena do disco e descamação da membrana fora com um pino de dissecação. Em seguida, execute imuno-histoquímica (passo 5.1.). </li></ol> 2. 50% pupação / Midpupal Retinas <ol><li> Segurar extremidade posterior do processo de pupa com um fórceps.Remover suavemente a superfície anterior do caso de pupa, agarrando a cutícula com outros fórceps e lentamente se afastar do pupário. Descascar cutícula residual para expor a cabeça. </li><li> Use as pontas dos fórceps &quot;para furar o saco, subjacente mole na região dorsal anterior. Isto permite o acesso à cabeça. Suavemente separar a cabeça do tórax por lentamente sugando a cabeça com uma pipeta P20. Expulsar cabeça de ponta da pipeta limpa e fria 1x PBS e remover o excesso de tecido; agarrar a cutícula cabeça e remover tromba. Prossiga para a etapa de fixação (passo 4.1.). </li><li> Pierce lóbulo da óptica com um pino para dissecar tecido estável. Inserir o outro pino de dissecação entre a lâmina e da retina para separar a retina a partir da lâmina / lóbulo óptico. Executar imunohistoquímica (passo 5.1.). </li></ol> 3. Olhos Adultos <ol><li> Pegue o centro da cápsula posterior da cabeça com uma pinça e remover partes da boca (tromba), TISS gorduraue e traquéia. </li><li> Agarre a cutícula cabeça dorsal com uma pinça e usar o outro para separar os olhos do tecido cerebral, puxando levemente para o lado. Na maioria dos casos, a lâmina, uma bainha fina e transparente de neuropil cérebro, vai ficar ligado à retina. É mais fácil remover a lâmina após a fixação. </li><li> Remover cutícula na margem do olho, mas deixam um pequeno pedaço trás para facilitar a transferência de retinas de soluções diferentes. Executar fixação (passo 4.1.). </li><li> Remover a lâmina, agarrando-o de um lado com uma pinça e puxando para o lado com a pinça outros sem prejudicar a retina. Remover cutícula residual, uma vez que pode dobrar na retina durante a montagem. Estes dois passos são críticos para a visualização de fotorreceptores, que de outro modo seria obscurecido pela lâmina e restos de cutícula durante o processo de criação de imagens. Proceder à imuno-histoquímica (passo 5.1.). </li></ol> 4. Fixação e Washing (cerca de 1 hora) <ol><li> Armazenar as retinas dissecadas em 1x PBS em um prato de vidro de três bem colocada em gelo durante a preparação do fixador fresco (diluído 40 microlitros de solução de formaldeído a 37% com 360 ul de PBS 1x, vórtice e armazenar em gelo). Recomendamos para prosseguir com o protocolo de fixação e para evitar tempos de armazenamento longos em gelo (&gt; 15 min). </li><li> Substituir 1x PBS com 200 ul de solução de formaldeído a 3,7%. Certifique-se de que o tecido é submergido em fixador. Isto é crítico, pois de outro modo não pode ser fixado correctamente. Se for necessário, remover as bolhas de ar e as traqueias com a pinça. </li><li> Incubar em agitador durante 15 min à temperatura ambiente. </li><li> Substituir fixador com 1x PBS e lavar duas vezes com PBS 1x. Continuar a lavagem em PBS durante 30 min (no agitador, à temperatura ambiente). </li><li> Substituir PBS com PBST. Lavar duas vezes com PBST. </li><li> Proceder com a remoção da membrana peripodial a partir de discos de olho larvais (3.1.3.) E as lâminas de midpupal / adulto retinas &lt;strong&gt; (3.2.3., 3.3.4.). </li></ol> 5. Imunohistoquímica <ol><li> Bloqueio: Substituir PBS com 200 ul de 5% de soro normal em PBST e retinas incubar durante 20 min em agitador à temperatura ambiente. </li><li> Substituir solução de bloqueio com uma solução de anticorpo primário. Selar o prato três bem com parafilm e incuba-se durante a noite no agitador à temperatura ambiente. </li><li> Substituir solução com PBST e lavar 3 vezes. Continuar a lavagem com PBST durante um mínimo de 1 hora no agitador à temperatura ambiente. </li><li> Transferência de solução de anticorpo secundário, e incubar em um prato de vidro selado parafilme bem no agitador durante a noite à temperatura ambiente. </li><li> Substituir solução de anticorpo com PBST e lavar três vezes. Continuar a lavagem com PBST durante um mínimo de uma hora no agitador à temperatura ambiente. As amostras podem ser mantidas em PBST durante vários dias, mas que é crucial para adicionar PBST fresco pelo menos uma vez por dia para evitar que sequem. </li></ol> 6. Montar <ol><li> Para a montagem de retinas larval e midpupal, utilize a P20 para transferir retinas para o centro de uma lâmina de vidro limpa. Remover o excesso de PBST. Use uma pinça para alinhar retinas, a fim de facilitar a criação de imagens. </li><li> Adicionar 10 ul de meio de montagem por cima das retinas. Cubra-os suavemente com um pedaço 24x40-1 de cobertura e vedação com unha polonês claro. </li><li> Para montagem retinas adultas, preparar &#39;pontes&#39; 20: Adicione duas gotas de 5 ul de glicerol a 50% em ambas as extremidades de um lado vidro e cobri-los com folhas de 22x22-1 de cobertura (0,13 a 0,17 milímetros de espessura). </li><li> Retire o PBST com uma pipeta P20, certifique-se de que as retinas não secar. Adicionar 20 ul de meio de montagem para o poço. Use meio de montagem para transferir retinas com o P20 para a lâmina de vidro &quot;ponte&quot;. </li><li> Use uma pinça para orientar (lentes deve estar virada para a lâmina de vidro) e alinhar retinas, a fim de facilitar a criação de imagens. Se for necessário, remover as bolhas de ar com o P20. </li><li> Lentamentedeslizamento lugar 24x40-1 cobertura (0,13 a 0,17 milímetros de espessura) de um lado em cima e as bordas de vedação com unha polonês claro ou fita adesiva. Armazenar as amostras a 4 ° C numa caixa de lâminas de microscópio saver (manter no escuro). </li></ol> 7. Resultados representativos Como exemplo para a aplicação deste protocolo, mostramos resultados representativos para visualizar a diferenciação de fotorreceptores nos três estágios de desenvolvimento no vídeo. Um disco de olho-antenais larval e uma retina midpupal foram corados com anticorpos que rotular diferentes tipos de fotorreceptores durante o seu desenvolvimento (Figura 1A, B) e um retina adulta foi corado com dois anticorpos rodopsina para visualizar dois subtipos de fotorreceptores (Figura 1C).

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1. Solução de problemas Em nossa experiência, dissecções requerem prática (várias semanas) e são facilitados por alcançar uma posição confortável da mão 21 por descansar os cotovelos e antebraços sobre a mesa e com os dedos fazendo contato com o prato dissecção. Dedos Dessa forma, apenas os polegares o índice e meio realizar movimentos sutis. Remover a lâmina sem danificar os fotorreceptores é provavelmente o passo mais desafiador. Prá...

<table border="1"><tr><td></td><td></td><td></td><td> Reagente </td></tr><tr><td> Salina tamponada com fosfato (PBS1x, pH 7,4) </td><td> Sigma </td><td></td><td> Preparar a solução estoque 10x 20. Dilui-se com água destilada para se obter 1 x PBS e armazenamento à temperatura ambiente. Esfriar em gelo antes de dissecações. </td></tr><tr><td> Triton-X 100 </td><td> Sigma </td><td> 9002-93-1 </td><td> Cuidado: Irritante Usar luvas. Preparar 50 ml de 1xPBS com 0,3% de Triton X-100 (PBST). Armazenar à temperatura ambiente. </td></tr><tr><td> Solução de formaldeído a 37% </td><td> Fisher Scientific </td><td> F75P1GAL </td><td> Cuidado: Tóxico, provável carcinógeno humano Usar luvas. Antes do passo de fixação, de fresco preparar uma solução 3,7% em uma capa química fumos por diluição com PBS, armazenar em gelo. </td></tr><tr><td> Soro de cavalo 5% normal </td><td> Jackson Immuno Research </td><td> 008-000-001 </td><td> Prepare a 5% de diluição em v / v em PBST. Armazenar em quatro graus. </td></tr><tr><td> Anticorpos primários e secundários (por exemplo, Donkey anti-carneiro Alexa Fluor 488, Donkey anti-coelho Alexa Fluor 555, Donkey anti-rato Alexa Fluor 647) </td><td> Sondas Invitrogen Molecular </td><td> A11015 A31572 A31571 </td><td> Diluir anticorpos secundários 1:800 em PBST e armazenar a quatro graus. </td></tr><tr><td> Alexa Fluor 488 Faloidina </td><td></td><td> A12379 </td><td> Diluir 1:100 em solução de anticorpo secundário. </td></tr><tr><td> Slowfade reagente Antifade Ouro </td><td> Sondas Invitrogen Molecular </td><td> S36936 </td><td> Meio de montagem. Armazenar a -20 graus. </td></tr><tr><td> Glicemiarol </td><td> Fisher Scientific </td><td> G31-1 </td><td> Para a montagem. Preparar 10 ml de diluição de 50% com água destilada armazenamento, à temperatura ambiente. </td></tr><tr><td> CO 2 </td><td></td><td></td><td> Para anestesiar moscas adultas. </td></tr><tr><td></td><td></td><td></td><td> Equipamento </td></tr><tr><td> Duas pinças afiadas (Dumont # 55) </td><td> Belas Science Tools </td><td> 11255-20 </td><td></td></tr><tr><td> Sylgard 184 Kit de elastômero de silicone </td><td> Dow Corning </td><td></td><td> Prepare Sylgard prato dissecção preenchendo uma placa de Petri de plástico com mistura Sylgard. </td></tr><tr><td> Três poços de dissecção pratos de vidro </td><td> Fisher Scientific </td><td> 21-379 </td><td></td></tr><tr><td> TwO pins minutien dissecando (0,1 mm de diâmetro) e dois pinholders (12 cm) </td><td> Belas ferramentas de Ciência </td><td> 26002-10 </td><td> Inserir um pino minutien em cada um dos dois pinholders e dobrar um dos pinos para formar um gancho. </td></tr><tr><td> Microscópio lamínulas (22x22-1 e 24x40-1) </td><td> Fisher Scientific </td><td> 12-542A </td><td></td></tr><tr><td> Lâminas de microscópio (25x75x1.0 mm; previamente limpas) </td><td> Fisher Scientific </td><td> 12-550-143 </td><td></td></tr><tr><td> Microcentrífuga 1,5 ml tubos </td><td></td><td></td><td></td></tr><tr><td> Limpar unha polonês ou fita adesiva </td><td></td><td></td><td></td></tr><tr><td> Orbital </td><td> Bélico </td><td></td><td></td></tr><tr><td> Caixa de suporte de slides </td&gt; <td> Fisher Scientific </td><td></td><td></td></tr><tr><td> Parafilm </td><td> Bemis </td><td> PM996 </td><td></td></tr><tr><td> Pincel fino </td><td></td><td></td><td></td></tr><tr><td> P20 e P200 micropipetas e dicas </td><td></td><td></td><td></td></tr><tr><td> Dissecando microscópio </td><td></td><td></td><td></td></tr><tr><td> Pequeno balde com gelo </td><td></td><td></td><td> Para o arrefecimento das placas de vidro assim, retinas dissecadas e soluções. </td></tr></table>

dissection and immunohistochemistry of larval, pupal and adult drosophila retinas

O olho composto de Drosophila melanogaster é composto por cerca de 750 omatídeos (olhos de unidades). Cada omatídeo é composto de cerca de 20 células, incluindo as células secretoras de lente de cone, as células de pigmento, uma célula de cerdas e oito fotorreceptores (RP) R1-R8 2. A RP têm estruturas especializadas microvilares, os rhabdomeres, que contêm pigmentos sensíveis à luz, os rodopsinas (RHS). Os rhabdomeres de seis PRs (R1-R6) formar um trapézio e conter Rh1 3 4. Os rhabdomeres de R7 e R8 são posicionadas em conjunto no centro do trapézio e partilhar o mesmo caminho de luz. R7 e R8 PRs estocasticamente expressar diferentes combinações de RHS em dois principais subtipos 5: Em subtipo &#39;p&#39;, RH3 em p R7s é acoplado com RH5 em p R8s, enquanto no subtipo do &#39;y&#39;, RH4 em y R7s está associada RH6 em y R8s 6 7 8. As primeiras especificações de RP e desenvolvimento de omatídeos começa no disco de olhos antenal larval imaginal, uma monocamada de células epiteliais. Uma onda de diferenciação varre o disco 9 e inicia a montagem de células indiferenciadas em omatídeos 10-11. R8 a &quot;célula fundador &#39;é especificada primeiro e recruta R1-6 e depois R7 12-14. Posteriormente, durante o desenvolvimento pupal, diferenciação PR leva a grandes mudanças morfológicas 15, incluindo a formação rhabdomere, sinaptogênese e expressão eventualmente rh. Neste protocolo, descrevemos métodos para dissecção da retina e imunohistoquímica em três períodos definidos de desenvolvimento da retina, o que pode ser aplicada para resolver uma variedade de questões relativas à formação da retina e vias de desenvolvimento. Aqui, usamos esses métodos para visualizar a diferenciação PR gradual no nível de uma única célula em toda montagem larval, midpupal e retinas adulto ( <sTrong&gt; Figura 1).

Watch this Scientific Journal Video about Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas at JoVE.com

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

O<em&gt; Drosophila</emRetina&gt; é uma estrutura cristalina do tipo composto por um pequeno número de tipos de células que são geradas de um modo estereotipado<sup&gt; 1</sup&gt;. Sua receptividade à análise genética sofisticada permite o estudo de complexos programas de desenvolvimento. Este protocolo descreve dissecações e imunohistoquímica de retinas em três distintas fases de desenvolvimento, com foco na diferenciação de fotorreceptores.

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

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

dissection-immunohistochemistry-larval-pupal-adult-drosophila

Research

JoVE Journal

Neuroscience

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

25.1K visualizações.  New York University. O Drosophila é uma estrutura cristalina do tipo composto por um pequeno número de tipos de células que são geradas de um modo estereotipado 1. Sua receptividade à análise genética sofisticada permite o estudo de complexos programas de desenvolvimento. Este protocolo descreve dissecações e imunohistoquímica de retinas em três distintas fases de desenvolvimento, com foco na diferenciação de fotorreceptores.

Vídeo: Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

Mapeamento e Aplicação de Enhancer-trap Expressão Flippase em larval e Adultos Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

Neurociência

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

Único Drosophila Dissection omatídeo e Imagem

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) 1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.
Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions 7, 8. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium 9, 10. Supporting cells are also important mediators of hair cell survival and death 11. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.

Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.

Dissecção da utrículo rato adulto e adenovírus Infecção mediada por células Coadjuvante

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear.  Since there ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

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

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

Live Imaging of Drosophila Larval Neuroblasts

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

Live Imaging of Drosophila Larval Neuroblasts

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

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

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

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of the nervous system often produce viable larvae and adults that have locomotion defective phenotypes that are difficult to adequately describe with text or completely represent with a single photographic image. Current modes of scientific publishing, however, support the submission of digital video media as supplemental material to accompany a manuscript. Here we describe a simple and widely accessible microscopy technique for acquiring high-quality digital video of both Drosophila larval and adult phenotypes from a lateral perspective. Video of larval and adult locomotion from a side-view is advantageous because it allows the observation and analysis of subtle distinctions and variations in aberrant locomotive behaviors. We have successfully used the technique to visualize and quantify aberrant crawling behaviors in third instar larvae, in addition to adult mutant phenotypes and behaviors including grooming.

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Here we describe a simple and widely accessible microscopy technique to acquire high-quality digital video of Drosophila adult and larval mutant phenotypes from a lateral perspective.

Aquisição de Vídeo de Alta Qualidade Digital de<em&gt; Drosophila</em&gt; Larvais e adultas Comportamentos de uma perspectiva lateral

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Monte todo Dissecção e Imunofluorescência do mouse cóclea Adulto

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Usando Canais Linear de agarose para o Estudo<em&gt; Drosophila</em&gt; Comportamento larval Crawling

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

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 ...

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 ...