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

NOTA: Todos los animales procedimientos fueron manejados en la Charité o Clínica Universitaria de Jena de acuerdo con las leyes de Cuidado Animal alemanes evitando cualquier sufrimiento innecesario de los animales. 1. Soluciones Preparación y Disección del lugar de trabajo <ol><li> Soluciones NOTA: Preparar las siguientes soluciones antes de comenzar la disección del epitelio. <ol><li> Soluciones para el procedimiento de disección: <ol><li> Preparar la solución de Ringer (pH 7,4) con concentraciones de NaCl 140 mM, KCl 5 mM, HEPES 10 mM, 2 mM CaCl 2, 1 mM de MgCl 2, y glucosa 10 mM. </li><li> Preparar una PBS - / - solución (pH 7,4) con concentraciones de 2.68 mM KCl, 1.47 mM KH 2 PO 4, NaCl 136 mM, y 8,1 mM Na 2 HPO 4. </li><li> Preparar una solución de fijación (pH 7,2) con concentraciones de 1x PBS - / -, 0,2 mM CaCl 2, 4% de sacarosa, y 4% de paraformaldehído. La sacarosa en la solución de fijación mejora crioseccionamiento y recoger de cryosections. Almacenar a -20 ° C. </li></ol></li><li> Soluciones para el procedimiento de tinción <ol><li> Preparar un PBS + / + solución con concentraciones de 1x PBS - / -, 0,48 mM MgCl2, 0,9 mM CaCl2. </li><li> Preparar una PBST + / + solución con concentraciones de 1x PBS + / + y 0,1% de Triton X-100. </li><li> Preparar una solución de bloqueo con concentraciones de 1x PBST + / + y 1% de gelatina. </li></ol></li></ol></li><li> Lugar de trabajo <ol><li> Para el lugar de trabajo de disección, usar un microscopio de disección con una iluminación brillante, así como una pluma líquido-bloqueante. </li><li> Preparar una placa de Petri, lleno de solución de Ringer, y portaobjetos de vidrio adhesivas. </li><li> Obtenga los siguientes instrumentos quirúrgicos: un par de tijeras quirúrgicas con un sostenido y una punta roma, un par de tijeras de primavera con forma de punta recta, dos pinzas con forma de punta curva fina y una hoja de afeitar ( <strong&gt; Figura 1A). </li></ol></li></ol> 2. Preparación del tabique nasal <ol><li> Casa animales en jaulas aprobados con acceso regular a alimentos y agua y el ciclo día / noche apropiada. </li><li> Realizar la anestesia en un recipiente que contiene una gasa empapada cerrado con 100% de isoflurano y el monitor analgesia probando los reflejos del pie trasero. Desde dislocación cervical puede causar que la sangre en las cavidades nasales, decapitar directamente ratones anestesiados. </li><li> Quitar la piel para exponer el hueso de todo el cráneo y la nariz, y limpiar la sangre y el tejido restante a fondo usando una toalla de papel. Retire la mandíbula inferior y los dientes delanteros. </li><li> Incisión en la médula dorsal de la nariz bilateralmente en 1-2 mm de distancia paralela a la línea de sutura para separar un lado del tabique (medial) de la maxilar (lateralmente). Dividir la nariz con un solo corte. Si los restos de hueso y cornetes todavía están unidos al tabique, eliminar estas cuidadosamente para exponer el tabique completamentesin tocarlo. </li><li> Quitar el hueso nasal dorsal deslizando la punta curvada multa de una pinza a lo largo de la cara dorsal del tabique, entre el septo y el hueso nasal. Aplique una ligera presión sobre el hueso, empuje hacia arriba y retírela. </li><li> Para proporcionar acceso a los dos tejidos septales, uno de cada cavidad, retirar cuidadosamente el maxilar superior del otro lado de la cabeza. En la preparación de los animales más viejos (P&gt; 28), retire la punta del hueso frontal que cubre los bulbos olfatorios. </li><li> Identificar el epitelio olfativo por su color ligeramente amarillo (Figura 1B). El epitelio respiratorio bordeando es blanco y muestra el movimiento de los cilios móviles que puede ser vista bajo el microscopio de disección. Corte a lo largo de la frontera entre el epitelio olfativo y respiratoria con un par de tijeras finas primavera. </li><li> Extienda el corte y separar el tabique de la conexión ventral al hueso vómer. A continuación, corte a lo largo de la frontera entre el tabique y la lámina cribosa del etmoides Ose. El tabique está completamente aislado de todas las conexiones a la cabeza. Use las posiciones de corte mostradas en la Figura 1C. </li></ol> 3. El aislamiento del epitelio olfativo para Inmunoticción <ol><li> Publicar un portaobjetos de vidrio adhesivo en la placa de Petri llena con solución de Ringer. Colocarlo sobre el borde de la placa de Petri, de manera que una mitad del vidrio se encuentra en solución de Ringer (Figura 1A). </li><li> Utilice unas pinzas para transferir cuidadosamente el hueso etmoidal con el epitelio olfativo a la placa de Petri. Levante sin agarrarlo con las puntas de las pinzas. </li><li> Coge la placa perpendicular del hueso etmoides con uno fórceps y utilizar un segundo uno para quitar cuidadosamente el epitelio de un lado del tabique. Deslice la punta curvada entre la lámina perpendicular y el epitelio de pelar el epitelio apagado. </li><li> Sé siempre asegúrese de identificar el lado ciliar, en caso de que el epitelio voltea en soluti de Ringeren. Si el epitelio voltea, no es posible identificar el lado ciliar después. Sobre todo, el epitelio se enrolla con la superficie ciliar dentro. </li><li> Comparar la forma de tejido inscrito con las posiciones de corte mostrado en la Figura 1C. Coge el epitelio en una posición en la frontera y tire de ella en el portaobjetos de vidrio. No toque el lado ciliar con las pinzas para evitar lesiones del tejido. </li><li> Girar el septum y repetir el procedimiento con el epitelio desde el otro lado. </li><li> Seque el portaobjetos de vidrio alrededor de las dos piezas de epitelio olfativo con una toalla de papel y rodear el tejido con un lápiz líquido bloqueador. </li><li> Fijar el tejido con la solución de fijación 150 l durante 10 min a RT. En cuanto a la estabilidad y la integridad de los cilios, usar tiempos de fijación cortos para una variedad de anticuerpos ensayados. Dentro de estos 10 min cilios son fijos, pero probablemente no todo el tejido epitelial. Por lo tanto, visualizar inmediata con el microscopio después de los scontienen procedimiento. Sin embargo, los tiempos pueden variar para la adherencia de anticuerpos individuales. </li></ol> 4. Protocolo de tinción NOTA: Maneje el tejido con mucho cuidado para preservar las estructuras ciliares de las neuronas sensoriales olfativas. Retire soluciones con la pipeta. No deje caer alguna solución directamente sobre el epitelio, como las fuerzas mecánicas podrían interrumpir la multa cilios. Realice todos los pasos a temperatura ambiente si no se indica lo contrario. <ol><li> Eliminar la solución de fijador y lavar 2 veces con PBS + / +. </li><li> Incubar el epitelio durante al menos 1 hora con solución de bloqueo. </li><li> Preparar la solución de anticuerpo primario mediante la disolución del anticuerpo en solución de bloqueo (1: 200 para los anticuerpos utilizados en este estudio) y centrifugar durante 5 min a velocidad máxima para eliminar cualquier precipitado. </li><li> Incubar el epitelio con una solución de anticuerpo en una cámara húmeda a 4 ° CO / N. </li><li> Epitelio lavado con PBS + / + durante 5 minutos al menos 3 veces. </li><li>Preparar la solución de anticuerpo secundario disolviendo el anticuerpo en solución de bloqueo (1: 500) y centrifugar durante 5 min a toda velocidad. </li><li> Incubar el epitelio con una solución de anticuerpo secundario durante 1 hr en una cámara oscura. </li><li> Epitelio lavado con PBS + / + durante 5 minutos al menos 3 veces. </li><li> Retire el líquido bloqueador con una hoja de afeitar o una toalla de papel. Lave el epitelio con agua destilada durante 5 s. </li><li> Preservar el tejido en antifade reactivo montaje. NOTA: La fluorescencia de fondo se incrementa rápidamente en cuestión de días; Por lo tanto, se recomienda el análisis microscópico, a más de 2 días después de la incorporación en medio de montaje. Especial cuidado se debe tener de no apretar el tejido cuando se busca el plano focal correcta, ya que puede dañar seriamente la preparación. Debido a la fluorescencia fuera de foco, se recomienda investigación adicional con microscopía 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...

El epitelio olfatorio del ratón en la cavidad nasal comprende 6-10000000 bipolares neuronas sensoriales olfativas 1. Cada neurona olfativa elige una de 1.200 genes de receptores odoríferos para la expresión. Detección de los odorantes comienza por la unión a un receptor olfativo 2, que entonces activa la adenilato ciclasa de tipo III (ACIII) 3 a través de la proteína G específica olfativa Ga olf 4 odorante. El consiguiente aumento de la adenosina monofosfato cíclico (cAMP) abre un nucleótido cíclico-cerrada (GNC), canal catiónico no selectivo que conduce a afluencia de Ca 2+ y Na +, y, posteriormente, Ca 2 + afluencia conduce a la apertura de un Cl Ca 2+ activado - canal de 5,6. El resultante hacia afuera Cl - flujo es facilitado por un alto Cl intracelular - concentración constante mantenida por Cl - absorción, probablemente a través de la Na + / K + / Cl - NKCC1 cotransportador, laCl - / HCO 3 - SLC4A1 intercambiador, y tal vez los transportistas adicionales aún por identificar 6-8. Neuronas olfativas bipolares tienen axones individuales, no ramificados que se proyectan directamente al bulbo olfatorio, y una dendrita que se extiende a la superficie del epitelio y termina como un compartimento especializado, el mando dendríticas. De este mando, 10-30 cilios, que puede alcanzar una longitud de hasta 50-60 micras, emanar en el moco que cubre la superficie epitelial 9. Las proteínas de la cascada de transducción de señal canónica se localizan principalmente en la membrana de estas cilios. El aumento de la superficie del epitelio sensorial amplifica la capacidad de detectar olores. Debido a la densidad de las neuronas sensoriales, cilios que se extiende desde las perillas dendríticas vecinos se entremezclan. Esta interrelación resultados en una mezcla aleatoria de los cilios de diferentes neuronas, que expresan diferentes tipos de receptores olfativos, en la superficie del epitelio. La detección y la célulaular asignación de las proteínas ciliares que sólo están presentes en un subconjunto de neuronas sensoriales tanto, es difícil en criosecciones. Además, la localización precisa de dichas proteínas a lo largo de los cilios es apenas posible, ya que cryosections son típicamente más delgada que la longitud media de los cilios. Para permitir la investigación de ciliar localización de las proteínas de membrana hasta ahora no caracterizados en las neuronas olfativas, hemos optimizado una técnica de preparación de la cara en la que permite el análisis detallado de la localización de proteínas en los cilios. En pocas palabras, se sacrifica el ratón y la cabeza dividió cerca de la línea media. Los cornetes, nasal, y los huesos frontales se retiran para exponer el tabique. El tabique con la parte olfativa del epitelio de revestimiento se afloja mediante la reducción de todas las conexiones a la cavidad nasal. Después de poner el tabique en una placa de Petri llena con solución de Ringer, el epitelio se despega und transferido a un portaobjetos de vidrio recubierto. Tras una breve fixatpaso de iones, procedimientos inmunotinción se puede realizar si la manipulación es tan suave como sea posible para evitar el daño del tejido frágil. Se demuestra la resolución alcanzable mediante la comparación de la tinción de dos proteínas de membrana diferentes en cilios olfatorios en cryosections clásicos y en la preparación cara es descrito.

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			<td style="width:127px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="42" style="height:42px;width:216px;">Spring scissors</td>
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			<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>
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		<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>
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		<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>
		</tr>
	</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.

Epitelio olfatorio en los preparativos de la cara puede ser utilizado para investigar la localización de las proteínas en los cilios de las neuronas sensoriales, lo que permite la investigación detallada de las proteínas cuya localización está claro después de un análisis de cryosections. Este problema puede ser ejemplificado en el caso de la tinción de la proteína para Kin-IRRE como 2 (Kirrel2). Kirrel2 (también llamado Neph3) es un miembro de la superfamilia de proteínas y funciones de la...

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.

Todo el montaje de etiquetado de los cilios en el sistema olfativo principal de ratones

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

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Neuroscience

11.0K vistas.  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.

Video: 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.

Visualización del sistema nervioso embrionario en Todo el montaje Drosophila Los embriones

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

Investigación

Neurociencias

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 fotólisis de compuestos enjaulados en los cilios de las neuronas sensoriales olfativas

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-etiquetado de las neuronas DRG para el Estudio de ramificación axonal en una preparación Todo el montaje del cable del ratón embrionario espinal

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.

El marcaje de células individuales en el Sistema Nervioso 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.

Todo el montaje de la tinción de inmunofluorescencia del Neonatal ratón Retina de investigar Angiogénesis<em&gt; En 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.

Disección Del Músculo Transverso Abdominis Para El Análisis De La Unión Neuromuscular De Montaje Completo

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.

Todo el montaje de Immunolabeling olfativa del receptor de las neuronas en el<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.

Imágenes en vivo de la ependimarios cilios en los ventrículos laterales del cerebro del ratón

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.

Todo el montaje La disección y de inmunofluorescencia del Adulto ratón cóclea

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 mediada por la expresión transgénica de constructos quiméricos para estudiar la función del comportamiento de los GPCR heterómeros en ratones

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