El proyecto descrito fue apoyada por número de concesión R01HD050725 del Instituto Nacional de Salud Infantil y Desarrollo Humano para etiquetar el contenido es de exclusiva responsabilidad de sus autores y no representa necesariamente las opiniones oficiales del Instituto Nacional de Salud Infantil y Desarrollo Humano o el Institutos Nacionales de Salud. Agradecemos a los miembros del laboratorio, así como Godenschwege Schreader Barbara por su aporte al manuscrito y video.

1. La disección de la cuerda ventral del nervio La disección y el tinte de llenado de la GF se puede realizar a temperatura ambiente. Para todos los pasos de la disección en frío (4 ° C) de solución salina se utiliza para disminuir el metabolismo celular y mantener las neuronas vivas por más tiempo. <ol><li> Anestesiar a las moscas adultas (4 días o más) con CO 2 o se ponen a enfriar en hielo hasta que se inmovilizan. Anestesia con hielo requerirá más tiempo (hasta 30 min), pero se mantienen las moscas inmovilizado durante más tiempo. </li><li> Coloque una mosca en un plato de Petri recubiertas Sylgard. Esta disección requiere el uso de un gran aumento (80-100X) estereomicroscopio disección. </li><li> Con unas tijeras Vannas, retire las patas, trompa y las alas. </li><li> El uso de fórceps finos y gruesos alfileres de insectos (pins minutien) para orientar la cara dorsal volar en el plato Sylgard. Pin el abdomen y luego se alargan un poco el animal mediante la colocación de dos pines en cada lado del cuello, detrás de la cabeza, de modo que el cuello es un poco estirado y accesibles (ver Figura 2). </li><li> Sumerja la marcha en una solución salina fría y fresca Drosophila. Más comúnmente utilizado salines Drosophila son suficientes para la disección, sin embargo aquí se utilizó una solución salina descrito en Gu y O&#39;Dowd, 2006 4. Una burbuja de aire que rodean la marcha. Extraer el aire con una jeringa. </li><li> Haga una pequeña incisión, lateral en el extremo dorsal del abdomen. A partir de la incisión, hacer un poco profundo, de corte recto a lo largo de la línea media dorsal del abdomen hacia el tórax. Luego continuar con el corte superficial, longitudinal en el tórax con la mayor precisión posible a lo largo de la línea media hacia el conectivo. Es muy importante para hacer los cortes superficiales para no dañar el VNC (Figura 1). </li><li> Use dos pernos de insectos muy bien para abrir cuidadosamente el tórax. Corre el cuerpo mitades con los pernos y coloque los pasadores a través de los músculos de vuelo (Figura 2). El tórax no se debe abrir aún más como se muestra en la Figura 2 para evitar la extracción de la cutícula. A fin de garantizar una orientación hacia el VNC los pines deben ser colocados en la misma posición anterior, posterior a cada lado. </li><li> El acceso a la VNC para la inyección de contraste y una imagen acústica correcta puede ser obstruida por los órganos internos. Por lo tanto, el uso de fórceps y tijeras para cortar el estómago, los intestinos, el corazón y los órganos reproductivos. Tire de las glándulas salivales tendido en ambos lados de la VNC. Enjuague la disección cuidadosa con solución salina para eliminar el tejido fresco flotantes y grasas. </li><li> Retire la grasa corporal por debajo de la conectivo cervical (CVC). La grasa corporal se ve mejor en un gran aumento. Use unas pinzas finas de entender cuidadosamente el tejido de la cara y tire de ella. </li></ol> La disección se puede realizar dentro de los 5 a 10 minutos. Mediante el uso de solución salina fría y fresca de la disección, el sistema nervioso en buen estado se mantendrá con vida durante al menos una hora. 2. Intracelular llenar GF Se recomienda realizar el tinte de llenado tan pronto como sea posible después de la disección. <ol><li> Coloque el plato Sylgard con la mosca disecados bajo un microscopio vertical, con un escenario fijo (Figura 3) y un objetivo de 10x de aire. Centro de la muestra con el extremo posterior de la mosca colocado hacia el soporte del electrodo llenar tinte. Use el contraste de interferencia diferencial (DIC) y el modo de enfoque en la CVC. Los axones grandes de la FC debe ser claramente visible en la superficie dorsal de la CVC. </li><li> Diluir Lucifer Amarillo CH sal de dilitio en H2O desionizada a una concentración del 1%. Tirar de un electrodo de vidrio borosilicato con filamento de una resistencia de aprox. 60-80 mW. La punta del electrodo de vidrio debe ser larga y afilada. Llenar la punta del electrodo con Lucifer solución al 1% Amarillo, colocando el electrodo con el extremo opuesto a la punta del electrodo en la solución de Lucifer amarillo durante aprox. 30-60 segundos. Luego rellenar el electrodo con 3M LiCl con una jeringa Hamilton dejando una burbuja de aire entre el colorante y LiCl. Conecte el electrodo de tinte de relleno para la etapa de la cabeza. </li><li> Inserte un alambre recubierto de cloruro de plata en el electrodo de tierra salina y colocar el electrodo de tinte llenar por encima del paralelo volar a la VNC utilizando un micromanipulador hidráulico 3D. El electrodo de llenar tinte debe ser conectado a un amplificador que permite que la corriente que pasa a fin de liberar positiva o negativamente a los tintes cargados desde el electrodo al pasar hiper o despolarizante actual, respectivamente. </li><li> Cambiar a una lente de 40x de inmersión en agua y baja la punta del electrodo de llenar tinte en una de las EFP. Los axones GF estaba cerca de la superficie dorsal de la CVC. Utilice una hacia adelante (hacia la cabeza), movimiento de insertar el electrodo en la GF. </li><li> Cambiar la fuente de luz a la luz de epifluorescencia y cambiar a un filtro amarillo Lucifer. Aplicar una corriente hiperpolarizante al electrodo de llenar tinte con el amplificador para inyectar el amarillo Lucifer en el GF. La cantidad de colorante inyectado puede ser controlado por thcantidad de corriente e pasado, así como la duración se pasa la corriente. Para evitar que la tinta se derrame fuera del sitio de la inyección o el daño del FG, se recomienda inyectar con una corriente baja (aproximadamente 3.5 nA) durante 1-5 minutos. </li><li> Aplicar actual hasta la terminal de GF en el VNC se puede ver claramente (Figura 4). Para trans-sináptica tinción de las neuronas postsinápticas, siguen paso de la corriente hasta el colorante también se puede ver en las neuronas postsinápticas. Desconecte la corriente y retire el electrodo llenar tinte. </li></ol> 3. Los resultados representativos: Un tinte representante de relleno para un GF con Lucifer amarillo se muestra en la Figura 4. La imagen fue tomada como una pila de imágenes en el software Nikon elementos con una cámara montada en el lugar microscopio. En esta corriente de la muestra se pasó a través del electrodo durante unos 2 minutos. El GF es completamente llena y la terminal de GF grande en el tórax es bien marcado (Figura 4, la elipse). No neuronas post-sinápticas son visibles en este caso, aunque Lucifer amarillo es lo suficientemente pequeño para pasar a través de uniones. Si el tinte no se inyecta el tiempo suficiente en el GF se trans-sináptica se llena de las neuronas postsinápticas pueden no ser vistos, porque la señal es demasiado débil para ser detectada. Neuronas postsinápticas de la FC (Figura 4B, puntas de flecha) se pueden visualizar con mayor fiabilidad el uso aún más pequeños marcadores neuronales, como Neurobiotin. La inyección de un Neurobiotin 7% en solución de acetato de 2 M de potasio con una corriente de despolarización durante 2 minutos es suficiente para calificar las neuronas postsinápticas con fuerza. Neurobiotin puede ser visualizado por microscopía confocal utilizando estreptavidina acoplada a los tintes de diferentes longitudes de onda. Porque Neurobiotin no es un tinte fluorescente, co-inyección de rodamina-Dextran o hidrazidas Alexa en la misma solución, se puede utilizar para controlar una inyección de éxito en el GF (Figura 4C, de Boerner y Godenschwege, 2010 5). <img src="/files/ftp_upload/3080/3080fig1.jpg" alt="Figura 1" /> Figura 1. Esquema de una mosca visto lateralmente. El tórax se muestra con una incisión longitudinal para visualizar las estructuras internas. Durante la disección, los músculos dorsales longitudinales de vuelo (DLMS, azul) se cortaron sin dañar el cordón nervioso ventral (VNC, de color rojo oscuro), que se encuentra ventral a los músculos de vuelo. El intestino (verde) se encuentra por encima de la VNC. <img src="/files/ftp_upload/3080/3080fig2.jpg" alt="Figura 2" /> Figura 2. Vista dorsal de una mosca disecada. Dos se colocan clavos detrás de la cabeza a la posición de la marcha. Los pines fijada a través de los músculos de vuelo (DLM) tienen el tórax abierto. Los órganos internos fueron trasladados a exponer el cordón nervioso ventral (VNC). <img src="/files/ftp_upload/3080/3080fig3.jpg" alt="Figura 3" /> Figura 3. Tinte de configuración de relleno. <img src="/files/ftp_upload/3080/3080fig4.jpg" alt="Figura 4" /> Figura 4. A) Z-proyección de la parte posterior de la VNC mostrando un GF lleno de Lucifer amarillo. La imagen fue tomada inmediatamente después de la tintura de relleno con una cámara de lugar. En animales de tipo salvaje de la terminal (elipse) en el tórax es claramente visible. B) la imagen confocal de un medio de GF se llenan de Neurobiotin en un vuelo de tipo salvaje. Tanto FC (flechas) y las neuronas postsinápticas de la FC (cabezas de flecha) están etiquetados. Neurobiotin se visualiza con estreptavidina acoplada a Cy3. C) la imagen confocal de tipo salvaje FC lleno de Alexa555.

<ol>
	<li>Power, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thoracico-abdominal+nervous+system+of+an+adult+insect+Drosophila+melanogaster.">The thoracico-abdominal nervous system of an adult insect Drosophila melanogaster.</a> J. Comp. Neurol. 88, 347-409 (1948).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, T. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+an+escape:+development+and+function+of+the+Drosophila+giant+fibre+system.">Making an escape: development and function of the Drosophila giant fibre system.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</li><li>Boerner, J., Duch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Average+shape+standard+atlas+for+the+adult+Drosophila+ventral+nerve+cord.">Average shape standard atlas for the adult Drosophila ventral nerve cord.</a> J. Comp. Neurol. 518, 2437-2455 (2010).</li><li>Gu, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+synaptic+transmission+in+adult+Drosophila+Kenyon+cells+in+sity.">Cholinergic synaptic transmission in adult Drosophila Kenyon cells in sity.</a> J. Neurosci. 26, 265-272 (2006).</li><li>Boerner, J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+for+the+Drosophila+ventral+nerve+cord+standard+in+neuronal+circuit+reconstruction+and+in-depth+analysis+of+mutant+morphology.">Application for the Drosophila ventral nerve cord standard in neuronal circuit reconstruction and in-depth analysis of mutant morphology.</a> J. Neurogent. 24, 158-1567 (2010).</li><li>Godenschwege, T. A., Kristiansen, L. V., Uthaman, S. B., Hortsch, M., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conderved+role+for+Drosophila+Neuroglian+and+human+L1-CAM+in+central-synapse+formation.">A conderved role for Drosophila Neuroglian and human L1-CAM in central-synapse formation.</a> Curr. Biol. 16, 12-23 (2006).</li><li>Uthaman, S. B., Godenschwege, T. A., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+distinct+from+highwire+for+the+Drosophila+ubiquitin+conjugase+bendless+in+synaptic+growth+and+maturation.">A mechanism distinct from highwire for the Drosophila ubiquitin conjugase bendless in synaptic growth and maturation.</a> J. Neurosci. 28, 8615-8623 (2008).</li><li>Storkebaum, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+mutations+in+the+tyrosyl-tRNA+synthetase+gene+recapitulate+in+Drosophila+features+of+human+Charcot-Marie-Tooth+neuropathy.">Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot-Marie-Tooth neuropathy.</a> Proc. Natl. Acad. Sci. USA. 106, 11782-11787 (2009).</li><li>Allen, M. J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+the+Drosophila+giant+fiber+system+(GFS).">Electrophysiological recordings from the Drosophila giant fiber system (GFS).</a> Cold Spring Harb. Protoc. 2010, pdb.prot5453-pdb.prot5453 (2010).</li><li>Augustin, H., Allen, M. J., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Recordings+from+the+Giant+Fiber+Pathway+of+D.+melanogaster.">Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster.</a> J. Vis. Exp. , (2011).</li></ol>

El sistema nervioso se puede diseccionar sin extraerlo del cuerpo volar. Esto tiene dos ventajas, en primer lugar, la disección causa poco daño al sistema nervioso, y en segundo lugar, la mayoría de los nervios permanecer conectados a los músculos y órganos sensoriales. Realización de la disección, como se describe, se prepara la muestra para el directo de tinte etiquetado de las finanzas públicas. Convenientemente, las motoneuronas postsináptica a la FC permanecer conectados a los músculos. Axones, por lo tan...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Sylgard 184 Kit de elastómero de silicona </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> Microscopio de disección </td><td> AmScope </td><td> SM-2TZ </td><td></td></tr><tr><td> Vannas tijeras superfinas </td><td> Instrumentos académicos </td><td> VS1023 </td><td></td></tr><tr><td> Dumont Pinzas Dumontstar 55 </td><td> Herramientas de Bellas Ciencia </td><td> 11295-51 </td><td></td></tr><tr><td> Austerlitz alfileres de insectos </td><td> Herramientas de Bellas Ciencia </td><td> 26002-10 </td><td> Ø 0,1 mm </td></tr><tr><td> Los electrodos de vidrio borosilicato </td><td> Mundial de Instrumentos de Precisión </td><td> 1B100F-4 </td><td></td></tr><tr><td> Extractor vertical con una pipeta de 700c </td><td> David Kopf Instruments </td><td> Modelo 700C </td><td></td></tr><tr><td> Lucifer Amarillo CH dilitio sal </td><td> Sigma </td><td> L0259 </td><td> 1% en H 2 O </td></tr><tr><td> Microscopio de fase fija vertical y cámara </td><td> Nikon </td><td> FN-1 y la cámara DS-U2 </td><td></td></tr><tr><td> Plan Fluor Objetivo de aire 10 veces </td><td> Nikon </td><td> CFI Plan Fluor 10X NA 0.3 16 mm WD </td><td></td></tr><tr><td> Agua Fluor 40x Objetivo de inmersión </td><td> Nikon </td><td> CFI Fluor 40x W NA 0.80 WD 2.0mm </td><td></td></tr><tr><td> Micromanipulador hidráulica 3D </td><td> Narishige Internacional </td><td> Modelo MW0-3 </td><td></td></tr><tr><td> Amplificador </td><td> Cómo Instruments, Inc. </td><td> Modelo 5A </td><td></td></tr></tbody></table>

whole mount preparation of the adult drosophila ventral nerve cord for giant fiber dye injection

Para analizar la morfología axonal y dendrítico de las neuronas, es esencial para obtener un correcto etiquetado de las estructuras neuronales. La preparación de muestras y etiquetado con poco o ningún daño a los tejidos nos permite analizar la morfología celular y para comparar las muestras individuales entre sí, permitiendo así la identificación de anomalías mutante. En el método de disección demostró que el sistema nervioso sigue siendo en su mayoría dentro de la mosca adulta. A través de una incisión dorsal, el abdomen y el tórax se abren y la mayor parte de los órganos internos son removidos. Sólo la parte dorsal del cordón nervioso ventral (VNC) y el conectivo cervical (CVC), que contiene los axones de las fibras grandes gigantes (FC) 1 se exponen, mientras que el cerebro contiene el cuerpo celular y las dendritas GF sigue siendo dos en la cabeza intacta . En esta preparación la mayoría de los nervios de la VNC debe permanecer unido a sus músculos. Después de la disección, el relleno intracelular de la fibra gigante (GF), con un tinte fluorescente se demuestra. En la CVC de los axones GF se encuentran en la superficie dorsal y por lo tanto pueden ser fácilmente visualizados con un microscopio con contraste de interferencia diferencial (DIC) óptica. Esto permite la inyección de los axones GF con el tinte en este sitio para la etiqueta del GF completo, incluyendo los axones y sus terminales en el VNC. Este método da lugar a manchas confiable y fuerte de la FC que permite a las neuronas para formar una imagen inmediatamente después del llenado con un microscopio de epifluorescencia. Por otra parte, la señal fluorescente se puede mejorar utilizando los métodos convencionales de inmunohistoquímica 3 adecuada para la microscopía confocal de alta resolución.

Watch this Scientific Journal Video about Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection at JoVE.com

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Un<em&gt; In vivo</em&gt; La disección de los adultos<em&gt; Drosophila</emCable&gt; nervioso ventral (VNC) se demuestra. Este método de disección en particular causa pocos daños en el VNC que permite el posterior marcado de las neuronas de fibra gigante con tinte fluorescente para obtener imágenes de alta resolución.

Preparación Todo el montaje del Adulto Drosophila Cordón nervioso ventral para inyección gigante tinte de fibra

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

whole-mount-preparation-adult-drosophila-ventral-nerve-cord-for-giant

Research

JoVE Journal

Neuroscience

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

18.9K vistas.  Florida Atlantic University. Un In vivo La disección de los adultos Drosophila nervioso ventral (VNC) se demuestra. Este método de disección en particular causa pocos daños en el VNC que permite el posterior marcado de las neuronas de fibra gigante con tinte fluorescente para obtener imágenes de alta resolución.

Video: Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

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

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

Registros electrofisiológicos de la ruta de fibra gigante de D. melanogaster</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Nanoinjection parejas y Ensayo de electrofisiología para la detección de bioactividad de compuestos utilizando el Drosophila melanogaster Sistema de fibra gigante

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Ratón reproducible aplastamiento del nervio ciático y posterior evaluación de la regeneración mediante el análisis de músculo entero Monte

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

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 ...

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 ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Un<em&gt; In Vitro</em&gt; Mouse Adulto Preparación músculo-nervio para estudiar las propiedades de cocción de los aferentes musculares

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

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 ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

La Preparación de oblicua de la médula espinal ventral de segmentos para Root Estimulación

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...