Este trabajo fue apoyado por una beca de Ehrman a HY. H., Jane Coffin Childs Memorial Fund para la beca de investigación postdoctoral médica a JJR, NIH subvención F32EY016309 a DV, una Beca Universidad de Nueva York Dean Disertación para DJ, NIH EY13010 GrantR01 para CD y una beca de la DFG a JR (RI 2208/1- 1). Damos las gracias a Nina Vogt y Boodram Pamela para comentarios sobre el manuscrito.

1. Introducción En este vídeo, se describen métodos para disecciones de retina y de inmunohistoquímica en tres períodos definidos de desarrollo: larvas del tercer instar, midpupal y la etapa adulta. Aunque nuestro protocolo también funciona para otros pupa (para más detalles sobre las etapas anteriores, véase 16), se optó por el escenario midpupal, ya que es óptima para todos los RP imágenes en un plano focal y sus núcleos son fácilmente identificables, lo que facilita la visualización de transcripción patrones de expresión del factor. La disección de los finales de retinas pupal es muy similar a la disección de retinas adultos. En primer lugar, se demuestra cómo recoger larvas y pupas en las etapas deseadas. A continuación, le explicamos cómo obtener disecciones óptimos de las larvas 17-18, midpupal retinas y adultos. En segundo lugar, demostramos cómo visualizar PR desarrollo en estas etapas utilizando microscopía inmunohistoquímica y confocal. El período de desarrollo de los insectos es dependiente de la temperatura. Para facilitar la puesta en escena reproducible de larvas y pupas, se recomienda levantar todas las poblaciones de la mosca a 25 ° C bajo un 12 h/12 ​​h luz / oscuridad ciclo. Larvas de tercer estadio: Sobre 96 horas después de la puesta de huevos (a 25 ° C), a finales de la alimentación de larvas de tercer estadio de parada y pasear en las paredes del vial de alimentos, donde eventualmente se convierten en crisálidas. Use un cepillo suave o un fórceps para extraer con cuidado un errante larva de tercer estadio de la pared del vial de alimentos. 50% de las pupas (&#39;midpupae&#39;) se observó aproximadamente 48 horas después de la pupación. Por el momento exacto, puedes encerrar pupas recién formadas blanco con un marcador en el frasco de comida. Alternativamente, se mueven todos pupas blanco a un nuevo vial para obtener un vial de estadificación uniforme (para la estadificación de pupas utilizando criterios morfológicos, ver 19). Después de 48 horas, con atención realove la midpupae del vial utilizando un fórceps. Las moscas adultas: anestesiar a las moscas adultas jóvenes (2-5 días después de la eclosión) en una plataforma de CO 2. Retire cabezales con un fórceps y almacenarlos en un vaso bien frío plato que contiene 1x PBS. 3. Procedimiento de Disección (aproximadamente 1-2 horas por prueba) Después de recoger la muestra, los coloca en una gota de PBS frío en un plato de disección Sylgard. Disecciona unos 10-15 retinas por genotipo. Por favor, consulte las hojas de seguridad, así como la salud ambiental de su institución y de la Oficina de Seguridad para la correcta manipulación de los reactivos y equipos (ver cuadros al final de este protocolo). 1. Los discos imaginales de larvas del ojo <ol><li> Localizar el extremo anterior de la larva, que contiene los ganchos de la boca. Utilice una pinza para presionar la parte media de la larva suavemente contra la base del plato Sylgard. Utilice Another fórceps para agarrar los ganchos de la boca y tirar suavemente y lentamente del cuerpo. Desechar la parte principal del cuerpo y el uso de las partes de la boca para mantener la parte anterior con el fin de eliminar el tejido extraño procedente de los discos imaginales que permanecen unidos al cerebro. </li><li> Traslado cerebro con los discos imaginales por el acaparamiento de los ganchos de la boca con una pinza en 1X PBS en una placa de vidrio de tres pozos. Realice el paso fijación (paso 4.1.). </li><li> Después de la fijación, eliminar las glándulas salivales, el tejido adiposo y tire del cerebro lejos del disco imaginal ojo-antenal. Para mejorar la accesibilidad de &quot;difíciles&quot; anticuerpos, la membrana peripodial, que se encuentra en la superficie del epitelio, se puede eliminar manteniendo presionada la parte antenal del disco y el pelado de la membrana de distancia con un pasador de disección. A continuación, lleve a cabo la inmunohistoquímica (paso 5.1.). </li></ol> 2. 50% pupación / Midpupal Retinas <ol><li> Mantenga el extremo posterior de la envoltura pupal con un fórceps.Retire con cuidado la superficie anterior de la envoltura pupal por el acaparamiento de la cutícula con otras pinzas y tire lentamente lejos de la pupa. Despegue la cutícula residual para exponer la cabeza. </li><li> Utilice las pinzas de puntas &quot;para perforar el saco subyacente, blando en la región dorsal anterior. Esto proporciona el acceso a la cabeza. Suavemente separar la cabeza del tórax por lentamente absorbiendo la cabeza con una pipeta P20. Expulsar a la cabeza de la punta de la pipeta en PBS frío fresco 1x y eliminar el exceso de tejido y consigue la cutícula y quite trompa. Continúe con el paso de fijación (paso 4.1.). </li><li> Pierce el lóbulo óptico con un pin de disección para estabilizar el tejido. Insertar el pasador de otro disección entre la lámina y la retina se separe la retina de la lámina / lóbulo óptico. Lleve a cabo la inmunohistoquímica (paso 5.1.). </li></ol> 3. Ojos adultos <ol><li> Coge el centro de la cápsula posterior de la cabeza con unas pinzas y quitar partes de la boca (probóscide), TISS grasaue y tráqueas. </li><li> Agarra la cabeza cutícula dorsal con un fórceps y utilizar la otra para separar los ojos de tejido cerebral tirando suavemente hacia los lados. En la mayoría de los casos, la lámina, una funda delgada y transparente de neurópilo cerebro, permanecerá unido a la retina. Es más fácil retirar la lámina después de la fijación. </li><li> Eliminar cutícula en el margen ojo, pero dejando un pequeño trozo detrás para facilitar la transferencia de retinas a diferentes soluciones. Realizar fijación (paso 4.1.). </li><li> Retirar la lámina por agarrarlo en un lado con un fórceps y tirando suavemente hacia los lados con los fórceps otros sin perjudicar la retina. Eliminar cutícula residual, ya que podría doblar sobre la retina durante el montaje. Estos dos pasos son fundamentales para la visualización de los fotorreceptores, que de otro modo quedar ocultas por la lámina y restos de cutícula durante el proceso de formación de imágenes. Proceda a la inmunohistoquímica (paso 5.1.). </li></ol> 4. La fijación y Washing (aprox. 1 hora) <ol><li> La tienda de las retinas disecadas en 1x PBS en un recipiente de vidrio de tres pozos se colocaron en hielo mientras recién preparar el fijador (diluir 40 microlitros de solución de formaldehído al 37% con 360 l de PBS 1x, mezclar y almacenar en hielo). Se recomienda proceder con el protocolo de fijación y para evitar largos tiempos de almacenamiento en hielo (&gt; 15 min). </li><li> Reemplazar 1x PBS con 200 l de solución de formaldehído al 3,7%. Asegúrese de que el tejido se sumerge en fijador. Esto es crítico, ya que de otra manera no se fija correctamente. Si es necesario, eliminar las burbujas de aire y las tráqueas con los fórceps. </li><li> Incubar en un agitador durante 15 minutos a temperatura ambiente. </li><li> Reemplazar fijador con 1x PBS y enjuague dos veces con 1x PBS. Continuar el lavado en PBS durante 30 min (en un agitador, a temperatura ambiente). </li><li> Reemplace PBS con PBST. Aclarar dos veces con PBST. </li><li> Proceder a la eliminación de la membrana de los discos peripodial ojos larvales (3.1.3.) Y las laminas de midpupal / adulto retinas &lt;strong&gt; (3.2.3., 3.3.4.). </li></ol> 5. La inmunohistoquímica <ol><li> Bloqueo: Reemplazar PBS con 200 l de 5% de suero normal en PBST y las retinas de incubar durante 20 min en un agitador a temperatura ambiente. </li><li> Sustituir solución de bloqueo con solución de anticuerpo primario. Sellar el plato de tres pozos con parafilm y se incuba durante la noche en un agitador a temperatura ambiente. </li><li> Reemplazar la solución con PBST y enjuague 3 veces. Continuar el lavado con PBST durante un mínimo de 1 hora en un agitador a temperatura ambiente. </li><li> Transferencia en solución de anticuerpo secundario e incubar en un plato de cristal Parafilm-sellado bien en un agitador durante la noche a temperatura ambiente. </li><li> Sustituir solución de anticuerpo con PBST y enjuagar tres veces. Continuar el lavado con PBST durante un mínimo de una hora en un agitador a temperatura ambiente. Las muestras se pueden mantener en PBST durante varios días, pero es crítico para añadir PBST fresco al menos una vez al día para evitar que se seque. </li></ol> 6. Montaje <ol><li> Para el montaje de retinas de larvas y midpupal, utilizar el P20 para transferir retinas al centro de un portaobjetos de vidrio limpio. Retire el exceso de PBST. Use una pinza para alinear retinas con el fin de facilitar formación de imágenes. </li><li> Añadir 10 l de medio de montaje en la parte superior de las retinas. Cubra ligeramente con una hoja de 24X40-1 tapa y selle con esmalte transparente. </li><li> Para el montaje retinas adultas, preparar &#39;puentes&#39; 20: Agregue dos gotas de 5 l de glicerol al 50% en ambos extremos de un lado del cristal y cubrir con cubreobjetos de 22x22-1 (0,13 a 0,17 mm de espesor). </li><li> Retire la PBST con una pipeta P20, asegúrese de que la retina no se sequen. Añadir 20 l de medio de montaje para el pozo. El uso medio de montaje para transferir retinas con la P20 a la diapositiva &quot;puente&quot; de vidrio. </li><li> Use una pinza para orientar (lentes deben mirar hacia la lámina de vidrio) y alinear retinas con el fin de facilitar la formación de imágenes. Si es necesario, elimine las burbujas de aire con el P20. </li><li> Despaciolugar 24X40-1 hoja de cubierta (0,13 a 0,17 mm de espesor) de un lado en la parte superior y los bordes de sellado con esmalte transparente o cinta adhesiva. Almacene las muestras a 4 ° C en una diapositiva de microscopio caja de ahorro (mantener en la oscuridad). </li></ol> 7. Los resultados representativos Como ejemplo de la aplicación de este protocolo, se muestran resultados representativos para la visualización de la diferenciación de los fotorreceptores en las tres etapas de desarrollo en el vídeo. Un ojo larval-antenal disco y una retina midpupal se tiñeron con anticuerpos que etiquetan los diferentes tipos de fotorreceptores durante su desarrollo (Figura 1A, B) y una retina adulta se tiñeron con dos anticuerpos rodopsina de visualizar dos subtipos fotorreceptoras (Figura 1C).

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1. Solución de problemas En nuestra experiencia, las disecciones requieren práctica (hasta varias semanas) y son facilitados por lograr una posición cómoda de la mano 21 descansando los codos y los antebrazos sobre la mesa y con los dedos en contacto con la placa de disección. De esta forma los dedos, los pulgares sólo el, índice y medio realizar movimientos sutiles. Extracción de la lámina sin dañar los fotorreceptores es probablemente el paso ...

<table border="1"><tr><td></td><td></td><td></td><td> Reactivo </td></tr><tr><td> Salina tamponada con fosfato (PBS1x, pH 7,4) </td><td> Sigma </td><td></td><td> Preparar una solución madre 10x 20. Diluir con agua destilada hasta obtener 1 x PBS y se almacena a temperatura ambiente. Enfriar en hielo antes de disecciones. </td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> 9002-93-1 </td><td> Precaución: Irritante Utilice guantes. Preparar 50 ml de 1xPBS con 0,3% Triton X-100 (PBST). Almacenar a temperatura ambiente. </td></tr><tr><td> 37% solución de formaldehído </td><td> Fisher Scientific </td><td> F75P1GAL </td><td> Precaución: carcinógeno tóxico, humano probable Utilice guantes. Antes de la etapa de fijación, recién preparar 3,7% de solución en una campana química diluyendo con PBS, tienda en el hielo. </td></tr><tr><td> 5% de suero normal de caballo </td><td> Jackson Immuno Research </td><td> 008-000-001 </td><td> Preparar 5% v / v en PBST dilución. Tienda a cuatro grados. </td></tr><tr><td> Anticuerpos primarios y secundarios (por ejemplo, burro anti-oveja Alexa Fluor 488, burro anti-conejo Alexa Fluor 555, de burro anti-ratón Alexa Fluor 647) </td><td> Invitrogen Molecular Probes </td><td> A11015 A31572 A31571 </td><td> Diluir secundaria de anticuerpos 1:800 en PBST y se almacena a cuatro grados. </td></tr><tr><td> Alexa Fluor 488 faloidina </td><td></td><td> A12379 </td><td> Diluir 1:100 en solución de anticuerpo secundario. </td></tr><tr><td> Slowfade Oro reactivo Antifade </td><td> Invitrogen Molecular Probes </td><td> S36936 </td><td> Medio de montaje. Almacenar a -20 grados. </td></tr><tr><td> Glycerol </td><td> Fisher Scientific </td><td> G31-1 </td><td> Para el montaje. Preparar 10 ml de 50% de dilución con agua destilada tienda, a la temperatura ambiente. </td></tr><tr><td> CO 2 </td><td></td><td></td><td> Para anestesiar a las moscas adultas. </td></tr><tr><td></td><td></td><td></td><td> Equipo </td></tr><tr><td> Dos pinzas cortantes (Dumont # 55) </td><td> Herramientas Artes Ciencias </td><td> 11255-20 </td><td></td></tr><tr><td> Sylgard 184 Elastómero de silicona Kit </td><td> Dow Corning </td><td></td><td> Preparar plato Sylgard disección llenando una placa Petri de plástico con la mezcla de Sylgard. </td></tr><tr><td> Tres pocillos de vidrio de disección </td><td> Fisher Scientific </td><td> 21-379 </td><td></td></tr><tr><td> Two minutien disección pins (0,1 mm de diámetro) y dos pinholders (12 cm) </td><td> Herramientas de Bellas Ciencia </td><td> 26002-10 </td><td> Insertar un pasador minutien en cada uno de los dos pinholders y doblar uno de los pasadores para formar un gancho. </td></tr><tr><td> Microscopio desliza la tapa (22x22-1 y 1-24X40) </td><td> Fisher Scientific </td><td> 12-542A </td><td></td></tr><tr><td> Portaobjetos (25x75x1.0 mm; limpieza previa) </td><td> Fisher Scientific </td><td> 12-550-143 </td><td></td></tr><tr><td> 1,5 ml tubos de microcentrífuga </td><td></td><td></td><td></td></tr><tr><td> Esmalte de uñas transparente o cinta adhesiva </td><td></td><td></td><td></td></tr><tr><td> Agitador orbital </td><td> Bellco </td><td></td><td></td></tr><tr><td> Slide cuadro titular </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 y P200 Micropipetas y puntas </td><td></td><td></td><td></td></tr><tr><td> Microscopio de disección </td><td></td><td></td><td></td></tr><tr><td> Pequeño cubo con hielo </td><td></td><td></td><td> Para el enfriamiento de las placas de vidrio, la retina disecados y soluciones. </td></tr></table>

dissection and immunohistochemistry of larval, pupal and adult drosophila retinas

El ojo compuesto de Drosophila melanogaster se compone de alrededor de 750 omatidios (ojos unitarios). Cada ommatidium se compone de alrededor de 20 células, incluyendo las células secretoras de cono-lente, células de pigmento, una célula de cerdas y ocho fotorreceptores (RP) R1-R8 2. Los BPs tienen estructuras especializadas microvillar, los rhabdomeres, que contienen pigmentos sensibles a la luz, la rodopsina (RHS). Los rhabdomeres de seis rupias (R1-R6) forman un trapecio y contienen Rh1 3 4. Los rhabdomeres de R7 y R8 están situados en tándem en el centro del trapezoide y comparte el mismo camino de la luz. R7 y R8 RP estocásticamente expresan diferentes combinaciones de RHS en dos subtipos principales 5: En el subtipo &#39;p&#39;, p RH3 en R7s se acopla con Th5 en p R8s, mientras que en el subtipo &#39;y&#39;, Th4 en R7s y se asocia con RH6 en y r8s 6 7 8. Especificación temprana de RP y desarrollo de omatidios comienza en el ojo larval-antenal disco imaginal, una monocapa de células epiteliales. Una ola de diferenciación barre a través del disco 9 e inicia el ensamblaje de células indiferenciadas en ommatidia 10-11. El R8 &#39;célula fundador se especifica primero y recluta R1-6 y R7 12-14. Posteriormente, durante el desarrollo pupal, PR diferenciación conduce a amplios cambios morfológicos 15, incluyendo la formación de rhabdomere, sinaptogénesis y la expresión final rh. En este protocolo, se describen métodos para disecciones de retina y de inmunohistoquímica en tres períodos definidos de desarrollo de la retina, que se pueden aplicar para abordar una variedad de cuestiones relativas a la formación de la retina y las vías de desarrollo. Aquí, nosotros usamos estos métodos para visualizar la gradual diferenciación PR a nivel de una sola célula en su totalidad larval monte, midpupal retinas y adultos ( <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

La<em&gt; Drosophila</emRetina&gt; es una red cristalina de tipo compuesto por un pequeño número de tipos de células que se generan de una manera estereotipada<sup&gt; 1</sup&gt;. Su docilidad al análisis genético sofisticado permite el estudio de complejos programas de desarrollo. Este protocolo describe disecciones e inmunohistoquímica de retinas en tres etapas de desarrollo discretos, con un enfoque en la diferenciación de los fotorreceptores.

La disección e inmunohistoquímica de larva, pupa y 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 vistas.  New York University. La Drosophila es una red cristalina de tipo compuesto por un pequeño número de tipos de células que se generan de una manera estereotipada 1. Su docilidad al análisis genético sofisticado permite el estudio de complejos programas de desarrollo. Este protocolo describe disecciones e inmunohistoquímica de retinas en tres etapas de desarrollo discretos, con un enfoque en la diferenciación de los fotorreceptores.

Video: 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.

La cartografía y la aplicación de potenciador de la trampa de expresión Flippase en larvas y adultos Drosophila SNC

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

Investigación

Neurociencias

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.

Solo Drosophila Omatidio disección e Imagen

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.

La disección de utrículo ratón adulto y mediada por adenovirus infección de reparto de las células

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 ...

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Drosophila Adulto olfativa Choque Aprendizaje

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including 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.

Imágenes en vivo de<em&gt; Drosophila</em&gt; larvales 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.

Adquisición de vídeo de alta calidad digital de<em&gt; Drosophila</em&gt; Comportamientos larvas y adultos desde una 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.

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 ...

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.

Uso de los canales lineales de agarosa para estudiar<em&gt; Drosophila</em&gt; Comportamiento de las larvas de arrastre

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.

Un simple de un solo paso para la disección Protocolo Todo el montaje Preparación de adulto<em&gt; Drosophila</emLos cerebros&gt;

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.

Análisis metabólico del cerebro adulto y Larval de Drosophila melanogaster

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