This work was funded by the Deutsche Forschungsgemeinschaft DFG (Exc257, SFB958).

NOTA: Todos os procedimentos com animais foram manipulados no Charité University Clinic Jena ou de acordo com as leis Animal Care alemães evitando qualquer sofrimento desnecessário dos animais. 1. Soluções preparação e Dissection Workplace <ol><li> Soluções NOTA: Prepare as soluções a seguir antes de iniciar a dissecção do epitélio. <ol><li> Soluções para o procedimento de dissecção: <ol><li> Preparar a solução de Ringer (pH 7,4) com concentrações de 140 mM de NaCl, 5 mM de KCl, 10 mM de HEPES, 2 mM de CaCl2, 1 mM de MgCl2, e 10 mM de glucose. </li><li> Preparar uma solução de PBS - / - solução (pH 7,4) com concentrações de 2,68 mM de KCl, 1,47 mM de Na KH 2 PO 4, 136 mM de NaCl, 8,1 mM e 2 HPO 4. </li><li> Prepara-se uma solução fixadora (pH 7,2) com concentrações de 1x PBS - / -, 0,2 mM de CaCl2, 4% de sacarose, e 4% de paraformaldeído. Sacarose na solução fixadora melhora crioseccionamento e pick-up de criosecções. Armazenar a -20 ° C. </li></ol></li><li> Soluções para o procedimento de coloração <ol><li> Preparar uma solução de PBS + / + solução com concentrações de 1x PBS - / -, 0,48 mM de MgCl2, 0,9 mM de CaCl2. </li><li> Prepara-se uma PBST + / + solução com concentrações de 1x PBS + / + e 0,1% de Triton X-100. </li><li> Prepara-se uma solução de bloqueio com concentrações de 1x PBST + / + e 1% de gelatina. </li></ol></li></ol></li><li> Local de trabalho <ol><li> Para o local de trabalho dissecção, usar um microscópio de dissecação com iluminação brilhante, bem como uma caneta líquido-bloqueador. </li><li> Prepara-se uma placa de Petri, cheia com solução de Ringer, e lâminas de vidro adesivas. </li><li> Obter os seguintes instrumentos cirúrgicos: um par de tesouras cirúrgicas com uma afiada e uma ponta romba, um par de tesouras de Primavera com forma de ponta de linha reta, duas pinças com ponta curvada forma fina e uma lâmina de barbear ( <strong&gt; Figura 1A). </li></ol></li></ol> 2. Preparação do Septo nasal <ol><li> Casa animais em gaiolas aprovados, com acesso regular a alimentos e água e do ciclo de dia / noite adequado. </li><li> Realizar anestesia em um recipiente contendo gaze embebida fechado com 100% de isoflurano e monitor de analgesia, testando reflexos nos pés traseiros. Desde deslocamento cervical pode causar sangue nas cavidades nasais, decapitar diretamente ratos anestesiados. </li><li> Retire a pele para expor o osso de todo o crânio e nariz, e limpar o sangue restante e tecido, cuidadosamente, com uma toalha de papel. Remover o maxilar inferior e os dentes da frente. </li><li> Incisão no osso dorsal do nariz bilateralmente em 1-2 mm de distância paralela à linha de sutura se separar um lado do septo (medial) da maxila (lateralmente). Dividir o nariz com um único corte. Se restos de ossos e conchas ainda estão ligados ao septo, retire-os cuidadosamente para expor o septo completamentesem tocá-lo. </li><li> Remover o osso nasal dorsal deslizando a ponta curvada de uma pinça fina ao longo do lado dorsal do septo, entre o septo nasal e osso. Aplique uma leve pressão sobre o osso, empurre-o para cima e remova-o. </li><li> Para proporcionar o acesso aos dois tecidos do septo, um de cada cavidade, remover cuidadosamente a maxila do outro lado da cabeça. Ao preparar os animais mais velhos (P&gt; 28), remova a ponta do osso frontal que cobre os bulbos olfatórios. </li><li> Identificar o epitélio olfactivo pela sua cor ligeiramente amarela (Figura 1B). O epitélio respiratório na fronteira é branco e mostra o movimento dos cílios móveis que podem ser vistas sob o microscópio de dissecação. Corte ao longo da fronteira entre o epitélio olfativo e respiratória com um par de tesouras de primavera. </li><li> Estender o corte e separar o septo a partir da conexão ventral ao osso vômer. Em seguida, corte ao longo da fronteira entre o septo e lâmina cribrosa do etmoidal Ose. O septo é agora completamente isolada de todas as conexões com a cabeça. Use as posições de corte mostrado na Figura 1C. </li></ol> 3. O isolamento do Olfativo Epithelium para Immunostaining <ol><li> Coloque uma lâmina de vidro adesiva na placa de petri cheia com solução de Ringer. Colocá-lo sobre o bordo da placa de petri, de modo que uma metade do vidro encontra-se em solução (Figura 1A) da campainha. </li><li> Use uma pinça para transferir cuidadosamente o etmóide com o epitélio olfativo à placa de Petri. Levantá-lo sem agarrando-o com as pontas fórceps. </li><li> Agarrar a placa perpendicular do osso etmóide com uma pinça e usar uma segunda para remover cuidadosamente o epitélio de um lado do septo. Deslize a ponta curvada entre a placa perpendicular e epitélio de descascar o epitélio off. </li><li> Seja sempre a certeza de identificar o lado ciliar, no caso de o epitélio vira em soluti do Ringerno. Se o epitélio vira, dificilmente é possível identificar o lado ciliar depois. Principalmente, o epitélio enrola com a superfície do ciliar dentro. </li><li> Comparar a forma de tecido inscritos com as posições de corte mostrado na Figura 1C. Agarre o epitélio em uma posição na fronteira e puxe-o para a lâmina de vidro. Não toque no lado do ciliar com a pinça para evitar lesões do tecido. </li><li> Ligue o septo e repetir o procedimento com o epitélio do outro lado. </li><li> Seca-se a lâmina de vidro em torno de ambas as peças de epitélio olfactivo com uma toalha de papel e cercam o tecido com uma caneta líquido-bloqueador. </li><li> Fixar o tecido com solução fixadora 150 uL durante 10 min à TA. No que diz respeito a estabilidade e integridade dos cílios, use tempos de fixação curtos para uma variedade de anticorpos testados. Dentro destes 10 min cílios são fixos, mas provavelmente não o todo tecido epitelial. Assim, visualizar imediato com o microscópio subsequente para os stenção procedimento. No entanto, os tempos de fixador pode variar para anticorpos individuais. </li></ol> 4. Coloração Protocol NOTA: Lidar com o tecido com muito cuidado para preservar as estruturas ciliares dos neurônios olfatórios. Remover soluções por pipetagem. Não deixe cair quaisquer soluções diretamente no epitélio, como forças mecânicas poderia interromper os cílios bem. Execute todas as etapas na RT se não for indicado o contrário. <ol><li> Remover a solução fixadora e lavar 2 vezes com PBS + / +. </li><li> Incubar o epitélio durante pelo menos 1 hora com solução de bloqueio. </li><li> Preparar a solução de anticorpo primário por dissolução do anticorpo em solução (1: 200 para os anticorpos utilizados neste estudo) de bloqueio e centrifugar durante 5 minutos a toda a velocidade para remover quaisquer precipitados. </li><li> Incubar o epitélio com solução de anticorpo numa câmara húmida a 4 ° CO / N. </li><li> Lavar epitélio com PBS + / + durante 5 minutos, pelo menos, 3 vezes. </li><li>Preparar a solução de anticorpo secundário por dissolução do anticorpo em solução (1: 500) de bloqueio e centrifuga-se durante 5 minutos à velocidade máxima. </li><li> Incubar o epitélio com solução de anticorpo secundário durante 1 h numa câmara escura. </li><li> Lavar epitélio com PBS + / + durante 5 minutos, pelo menos, 3 vezes. </li><li> Retirar o líquido-bloqueador com uma lâmina de barbear ou toalha de papel. Lava-se a epitélio com água destilada durante 5 s. </li><li> Preserve o tecido em antifade reagente de montagem. NOTA: fluorescência de fundo aumenta rapidamente dentro de dias; análise microscópica, o mais tardar dois dias após a incorporação no meio de montagem é, portanto, recomendada. Cuidados especiais devem ser tomados para não apertar o tecido ao pesquisar o plano focal correto, uma vez que pode causar sérios danos à preparação. Devido a fluorescência fora de foco, é recomendável uma investigação mais aprofundada com a microscopia confocal. </li></ol>

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The authors have nothing to disclose.

The en face preparation technique described in this protocol provides a powerful tool for the detailed analysis of the olfactory system. So far, most studies characterizing the localization of signaling proteins use immunostainings of cryosections. They present a good overview of the olfactory epithelium, and protein expression in distinct cell types or regions can be easily identified. However, expression in olfactory cilia is sometimes hard to detect. Even if ciliary localization is obvious, cryosections offer...

O epitélio olfativo do mouse na cavidade nasal compreende 6-10.000.000 neurônios olfatórios bipolares 1. Cada neurônio olfativo escolhe um dos 1.200 genes de receptores olfativos para expressão. Detecção de odorantes inicia por ligação a um receptor olfactivo 2, que, em seguida, activa adenilil ciclase do tipo III (ACIII) 3 através da proteína G específica olfactiva Gu olf 4 odorante. O aumento resultando em monofosfato de adenosina cíclico (cAMP) abre uma cíclico (CNG), canal de cátions não seletivos levando a influxo de Ca 2+ e Na +, e, posteriormente, Ca 2+ fechado de nucleotídeo influxo leva à abertura de um Cl Ca 2+ ativado - canal 5,6. O resultante exterior Cl - fluxo é facilitada por uma alta Cl intracelular - concentração mantida constante por Cl - absorção, provavelmente através de Na + / K + / Cl - NKCC1 co-transportador, oCl - / HCO3 - trocador SLC4A1, e transportadores ainda a ser identificados talvez adicionais 6-8. Neurónios olfactivos bipolares têm axónios, não ramificados individuais que se projectam directamente para o bolbo olfactivo e um dendrito que se estende para a superfície do epitélio e termina como um compartimento especializado, o botão dendrítica. A partir deste botão, 10-30 cílios, que pode atingir um comprimento de até 50-60 mm, emanam no muco que cobre a superfície epitelial 9. As proteínas da cascata de transdução de sinal canónico estão principalmente localizados na membrana destas cílios. O aumento da superfície do epitélio sensorial amplifica a capacidade de detectar odorantes. Devido à densidade de neurônios sensoriais, os cílios se estende desde maçanetas dendríticas vizinhos se misturam. Este entrelaçamento resulta numa mistura aleatória dos cílios de diferentes neurónios, que expressam diferentes tipos de receptores olfactivos, na superfície do epitélio. A célula de detecção ealocação ular de proteínas ciliares que estão presentes em apenas um subconjunto de neurônios sensoriais, portanto, difícil de criosecções. Além disso, a localização precisa de tais proteínas ao longo dos cílios é quase impossível, visto que criocortes são tipicamente mais fina do que o comprimento médio dos cílios. Para ativar a investigação da ciliar localização de proteínas de membrana até agora descaracterizados em neurônios olfativos, otimizamos um en face preparação técnica que permite a análise detalhada de localização de proteínas em cílios. Resumidamente, o rato é sacrificado ea cabeça dividida perto da linha média. Cornetos nasais, e os ossos frontais são removidos para expor o septo. O septo com a parte olfativa do epitélio de revestimento é solto por cortar todas as conexões para a cavidade nasal. Depois de colocar o septo numa placa de petri cheia com solução de Ringer, o epitélio é descascada und transferido para uma lâmina de vidro revestido. Após uma breve FIXATion passo, os procedimentos imunocoloração pode ser realizada se a manipulação é o mais suave possível para evitar danos do tecido frágil. Nós demonstramos a resolução alcançável através da comparação da coloração de duas proteínas de membrana em diferentes cílios olfactiva em criocortes clássicos e na preparação cara en descrito.

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			<td style="width:127px;">Company</td>
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			<td style="width:167px;">Comments/Description</td>
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			<td height="42" style="height:42px;width:216px;">Spring scissors</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">straight tip, multiple suppliers</td>
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			<td height="42" style="height:42px;width:216px;">Surgical scissors</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">sharp and blunt end, multiple suppliers</td>
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			<td height="42" style="height:42px;width:216px;">Fine forceps</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">curved tips, Dumont #7, multiple suppliers</td>
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			<td height="42" style="height:42px;width:216px;">Razor blade</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">extra thin, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Binocular with illumination</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiple suppliers, Stemi 2000-C, Zeiss</td>
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			<td height="21" style="height:21px;width:216px;">Petri dish</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiple suppliers</td>
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			<td height="21" style="height:21px;width:216px;">Liquid-blocker pen</td>
			<td style="width:127px;">Science Services</td>
			<td style="width:119px;">N71310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polysine coated slides</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:119px;">J2800AMNZ</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">Confocal microscope</td>
			<td style="width:127px;">Leica Microsystems</td>
			<td style="width:119px;">TCS SPE</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary antibody Goat anti-Kirrel2</td>
			<td style="width:127px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2930</td>
			<td style="width:167px;">1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary antibody Rabbit anti-mOR-EG</td>
			<td style="width:127px;">Baumgart et al., 2014</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">secondary antibodies</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">A21206, A11057</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mounting medium, ProLong Gold antifade reagent</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">P36930</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paraformaldehyde</td>
			<td style="width:127px;">Sigma</td>
			<td align="right" style="width:119px;">441244</td>
			<td style="width:167px;">toxic, work under fume hood</td>
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	</tbody>
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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.

Epitélio olfactivo en preparações cara pode ser utilizado para investigar a localização de proteínas nos cílios dos neurónios sensoriais, o que permite a investigação aprofundada das proteínas cuja localização é claro após análise de criocortes. Este problema pode ser exemplificado no caso da coloração de proteína para Kin IRRE-like 2 (Kirrel2). Kirrel2 (também chamado Neph3) é um membro da superfamília de imunoglobulina (Ig) de proteínas e funções da membrana como uma proteín...

Watch this Scientific Journal Video about Whole Mount Labeling of Cilia in the Main Olfactory System of Mice at JoVE.com

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

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 ...

whole-mount-labeling-of-cilia-in-the-main-olfactory-system-of-mice

Research

JoVE Journal

Neuroscience

11.0K visualizações.  Universitaetsklinikum Jena. 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.

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

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

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Flash Fotólise de Compostos Caged no Cilia de Olfactory neurônios sensoriais

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII-rotulagem de neurônios DRG para estudar em uma ramificação axonal Preparação Monte Whole of Mouse Embryonic Spinal Cord

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

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

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

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

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

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

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

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Whole Mount coloração de imunofluorescência do Rato Retina Neonatal para investigar angiogênese<em&gt; In vivo</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Dissecção do Músculo Transversus Abdominis para Análise de Junção Neuromuscular de Montagem Integral

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

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

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

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

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

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

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Imagens ao vivo da ependimária Cilia nas laterais ventrículos do cérebro do rato

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

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 ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

HSV-Mediated expressão do transgene da quiméricos Construções para estudar a função comportamental de GPCR heterómeros em Ratos

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...