Reconocemos el centro de Centro de Recursos de Drosophila Viena y Bloomington Drosophila (NIH P40OD018537) para proporcionar cepas de Drosophila. Agradecemos a Jack Fransen desde el Centro de Imagen microscópica de apoyo de expertos en imágenes. Este estudio fue apoyado por becas VIDI y superior (917-96-346, 912-12-109) de la Organización Holandesa para la Investigación Científica (NWO), por dos becas de doctorado / Universidad de Radboud DCN Medical Center, por el Retraso Mental Red Alemana financiado por el programa NGFN + del Ministerio Federal alemán de Educación e Investigación (BMBF) y por Gencodys de la Unión Europea 7PM gran escala integrados de red (SALUD-241995) a AS. Los donantes no tenía papel en el diseño del estudio, la recogida y análisis de datos, decisión a publicar, o la preparación del manuscrito.

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Los autores no tienen conflictos de intereses a revelar.

&quot;Drosophila NMJ Morfometría&quot; y &quot;Drosophila NMJ Bouton Morfometría&quot; son herramientas poderosas para los investigadores interesados en la evaluación de la morfología de la sinapsis. evaluación manual de los parámetros NMJ es laborioso; se estima que las macros se ahorrarían un investigador experimentado hasta 15 min / NMJ pasó en la segmentación manual de imagen. Con una o dos docenas de sinapsis evaluados por condición o genotipo, esto resume rápidamente hasta una cantidad considerable de tiempo ahorrado, incluso en estudios a pequeña escala. Al realizar grandes pantallas, la ganancia de la utilización de análisis de alto rendimiento, en comparación con la evaluación manual y la cuantificación, puede ser inmensa. Además de un mayor rendimiento, los macros proporcionan fácilmente análisis objetivo; excluyen a los prejuicios personales que de otra forma requieren experimentos ciegos, así como las diferencias interpersonales que se producen cuando varios investigadores participan en el análisis. Por último, las macros proporcionan una una sensible y precisoÁLISIS de características NMJ, lo que permite la identificación de reguladores sinápticas que causan más sutil que defectos NMJ dramáticos y hasta ahora han permanecido poco apreciado por el ojo del investigador. La información detallada sobre los procedimientos de validación y los algoritmos utilizados en las macros se encuentran en la publicación Nijhof et al. 17. La funcionalidad de los macros se ha validado para medir apropiadamente características morfológicas de Drosophila melanogaster NMJs en músculo 4. Posteriormente, se demostró que las macros también eran adecuados para analizar las sinapsis en otros músculos en este organismo. Es probable que las macros también se pueden usar para medir los parámetros morfológicos de NMJ con estructura similar en otras especies, incluyendo otras especies de Drosophila y otros insectos. Incluso NMJs muy distantes en la evolución, por ejemplo, NMJs de ratones, muestran una conformación estructural bastante similar 29. Las macros no se han probado en los preparativos NMJ de otras especies, pero los usuarios potenciales se les anima a probar las macros para tales fines. Es muy importante que el usuario explora los diferentes umbrales de automóviles y algoritmos para definir / seleccionar los ajustes de macro más adecuados para las imágenes. Con estos ajustes, se logra una precisión de aproximadamente el 95% cuando la evaluación macro se comparó con la evaluación manual. Ajuste de los valores de la macro a adecuadamente segmento 100% de las imágenes puede ser un procedimiento muy laborioso o incluso imposible. Por lo tanto, la exclusión de las imágenes no segmentados correctamente se recomienda si su número es inferior al 5%. Evidentemente, si la calidad de las imágenes es baja, las macros se generan mayores proporciones de segmentaciones imagen insatisfactorios. Las imágenes de baja calidad influirán de manera similar la evaluación manual y por lo tanto no pueden ser ligada a la evolución de las macros. No obstante las macros son bastante robusta ya que fueron diseñados para una imagenS generada en un microscopio de alta contenido (un microscopio de fluorescencia automatizado que permite obtener imágenes de gran número de muestras) 17. Un punto crítico es que el usuario inspecciona visualmente todas las imágenes resultado generado por las macros. Esto permitirá detectar y excluir las imágenes con la segmentación insatisfactorio. En la sección 6 de este protocolo, el usuario es guiado cómo ajustar la configuración de la segmentación de imagen correcta cuando se ejecuta el sub-macro &quot;Analizar&quot;. Para familiarizarse rápidamente con los requisitos de las macros y cómo ajustar los valores de la macro una carpeta llamada &quot;Examples_adjusting valores de la macro&quot; está incluido en el repositorio de https://figshare.com/s/ec634918c027f62f7f2a macro. Trece subcarpetas, cada uno con ejemplos de imágenes obtenidas en diferentes plataformas de microscopio (microscopios alto contenido / confocal / fluorescencia) y diferentes immunostainings, se proporcionan. Un PDF titulado “guía Ejemplos” se incluye en el mismocarpeta en la que se proporcionan los ajustes requeridos para cada ejemplo, junto con un documento de texto que proporciona los resultados esperados y los resultados de las imágenes. Las macros se han diseñado para procesar las imágenes guardadas como archivos separados .tiff, sin embargo, algunos usuarios podrían haber salvado sus imágenes en un formato diferente. La siguiente página web https://figshare.com/s/ec634918c027f62f7f2a 21 contiene una carpeta con el nombre &quot;Drosophila NMJ&quot; donde tres archivos de ejemplo (Ejemplo 1 - 3) y la &quot;Guía de ejemplos&quot; de documentos con instrucciones detalladas de cómo importar imágenes en la macro si no se almacenan como archivos separados .tiff también se pueden encontrar en la misma carpeta. En conjunto, las macros &quot;Drosophila NMJ Morfometría&quot; y &quot;Drosophila NMJ Bouton morfometría&quot; cuantificar diez diferentes características NMJ: área NMJ, perímetro NMJ, número de boutons, área Bouton NMJ, longitud NMJ, NMJ más larga que la longitud de la rama, número de IslaNDS, número de ramas, el número de puntos de ramificación y el número de zonas activas. Esto proporciona una gran ventaja sobre las herramientas disponibles hasta ahora que pueden evaluar sólo una o algunas de sus funciones sinápticas 30, 31. Análisis cuantitativo Multiparámetrico lleva un gran potencial para nuevos descubrimientos, por ejemplo, para identificar nuevos reguladores que controlan uno hasta numerosos aspectos de la biología sinapsis. También proporciona la resolución necesaria para determinar los genes que correguladores de exactamente las mismas o superposición de las características NMJ y por lo tanto es probable que operar en vías moleculares comunes. Por último, se abre la posibilidad de investigar cómo los diferentes parámetros sinápticos se correlacionan entre sí en condiciones inalteradas 17 y aseguran que los genes tales correlaciones morfométricos coordinados. En su conjunto, este protocolo se ilustra cómo utilizar las dos macros &quot;Drosophila NMJ Morfometría&quot; y&quot;Drosophila NMJ Bouton Morfometría&quot;, que realizan la cuantificación objetiva y sensible de diez características morfológicas NMJ en una forma de alto rendimiento.

Trastornos cognitivos tales como la discapacidad intelectual, trastorno del espectro autista y la esquizofrenia se caracterizan a menudo por una función anormal sináptica 1, 2, 3. morfología Synapse y función están estrechamente entrelazadas; defectos morfológicos pueden causar un mal funcionamiento sináptica y, a la inversa, la transmisión sináptica aberrante impactarán la maduración sináptica y la morfología 4, 5, 6. Una serie de organismos modelo se han empleado con el fin de entender mejor la biología de la sinapsis y arrojar luz sobre cómo los cambios sinápticos afectan la función cerebral en salud y enfermedad 7, 8, 9. La Drosophila NMJ es una extensamente estudiada y bien establecido modelo in vivo para sy glutamatérgicanapse biología 10, 11. En las últimas décadas, este modelo se ha utilizado para los estudios fisiológicos y centrado genes, así como para las pantallas de genética a gran escala, con el objetivo de detectar diferencias morfológicas entre NMJs. En particular, las pantallas adelante genéticos han identificado muchos reguladores cruciales y mecanismos del desarrollo de sinapsis y la función 12, 13, 14, 15, 16. Sin embargo, la mayoría de estas pantallas se basó en la evaluación visual de la NMJ morfología terminal y en la detección cualitativa de las anormalidades sinápticas o de puntuación semicuantitativa de algunas características morfológicas. Como consecuencia, más sutiles alteraciones morfológicas sinápticas que no son obvias para el ojo humano se pierden fácilmente. Con el fin de poder detectar diferencias cuantitativas forma integral, lasNMJ tiene que ser evaluada con precisión mediante la cuantificación sistemática de los parámetros morfológicos de interés. La medición de características NMJ manual es laborioso, especialmente cuando hay varias características NMJ de interés y / o cuando se realizan pruebas de detección genéticos a gran escala. Para apoyar multiparamétrica, análisis morfológico de alto rendimiento y para lograr la cuantificación objetiva, dos macros &quot;Drosophila NMJ morfometría&quot; y &quot;Drosophila NMJ Bouton morfometría&quot; se desarrollaron 17. Ambas macros se ejecutan en el software de código abierto de análisis de imágenes Fiji 18, y pueden cuantificar ambas imágenes confocal y nonconfocal. Medidas &quot;Drosophila NMJ morfometría&quot; terminales NMJ inmunoteñidas con el disco postsináptica marcador de gran-1 (Dlg-1) o la peroxidasa de rábano picante presináptica (HRP), co-etiquetados con el Bruchpilot marcador de zona activa (BRP). Cuantifica nueve paráme morfológicaTER (descrito más adelante): área NMJ, perimetrales NMJ, número de boutons, longitud NMJ, NMJ más larga longitud de rama, número de islas, número de ramas, número de puntos de ramificación, el número de zonas activas en la terminal sináptica (Figura 1) . Aunque un algoritmo para determinar el número de boutons está presente en esta macro, que no cumplía con los criterios de exactitud 17. Para evaluar adecuadamente el número de boutons, es necesario utilizar la macro &quot;Drosophila NMJ Bouton Morfometría&quot;, que está diseñado específicamente para cuantificar boutons utilizando preparaciones NMJ inmunoteñidas por anti-sinaptotagmina (Syt) o proteína de cadena anti-Cisteína (Csp), y co-immunolabeled con Brp. La macro &quot;Drosophila NMJ Bouton Morfometría&quot; cuantifica los siguientes parámetros: número de boutons, área Bouton NMJ, longitud NMJ, NMJ más larga longitud de la rama, número de islas, número de ramas, el número de puntos de ramificación y el número de zo activones (Figura 2). Las macros consisten en 3 sub-macros: (I) &quot;Convertir a apilar&quot; identifica todos los archivos de imagen disponibles y crea Z-hyperstacks y proyecciones de máxima intensidad de ambos canales. Como salida, esta macro generará dos nuevos archivos por sinapsis llamada &quot;stack_image_name&quot; y &quot;flatstack_image_name&quot;. II) &quot;Definir ROI&quot; se abrirá todas las imágenes de proyección máximos &quot;flatstack_image_name&quot; consecutivamente y presentarlos con la solicitud para definir manualmente la región de interés (ROI) en el que está presente el terminal sináptica específica de interés. Esto se implementó para permitir la exclusión de las sinapsis que conectan a los músculos adyacentes y / u otros tipos de terminales sinápticas (tales como 1s) que pueden estar presentes en las imágenes 11. (III) &quot;Analizar&quot; se aplica totalmente automatizado análisis a todas las regiones de las imágenes dentro de las fronteras de la ROI. Comoresultado de este paso, el usuario obtendrá dos nuevos archivos: &quot;results.txt&quot;, donde se anotarán toda la medición numérica y un &quot;res_image_name.tif&quot;, donde se ilustrarán las segmentaciones imagen subyacente producidos por la macro. Durante el análisis de imagen tres estructuras se derivan de cada terminal sináptica: el contorno NMJ, el esqueleto NMJ, y el número de zonas activas Brp-positivos. El contorno NMJ se utiliza para determinar el área NMJ y su perímetro y una separación de cuencas posterior proporciona el número de boutons. Desde el esqueleto, cinco características NMJ se deducen: la longitud total NMJ, la suma de la longitud de la trayectoria continua más larga que conecta dos puntos finales (la más larga longitud de la rama), el número de compartimentos no conectados por NMJ (referido como &quot;islas&quot; ), el número de ramas, y el número de puntos de ramificación (un punto de ramificación conecta tres o más ramas). El número de zonas activas se determina en el Brp canales contandomanchas BRP-positivas. El esquema anotado NMJ (línea amarilla), el esqueleto NMJ (línea azul), y el número de zonas activas Brp-positivos (indicados por focos blanco) se visualizan en un cuadro los resultados y las medidas de los parámetros se procesan a una (. txt) archivo de salida (Figura 3). Drosophila NMJ Morfometría&quot; y &#39;Drosophila NMJ Bouton Morfometría&#39; se describieron primero y extensamente validados por Nijhof et al. 17. Este manuscrito se centra en la metodología de análisis de la morfología NMJ usando las macros &#39;Drosophila NMJ morfometría&#39; y &#39;Drosophila NMJ Bouton morfometría&#39;. Sin embargo, antes de los análisis macro-asistida, disecciones NMJ y immunostainings deban llevarse a cabo. Estos son pasos cruciales, y la combinación de marcadores utilizados para inmunohistoquímica tiene que ser adecuado para los análisis de macro. Estos pasos se mencionan brevemente en sEcción 1 de este protocolo y dirigir al usuario a referencias que describen en detalle los protocolos para ejecutar estos procedimientos.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Immunostainings</td>
			<td></td>
			<td></td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Mouse anti-discs large 1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>AFFN-DLG1-4D6</td>
			<td>1/25 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit)</td>
		</tr>
		<tr>
			<td>Rabbit anti-horseradish peroxidase</td>
			<td>Jackson IR</td>
			<td>323-005-021</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Rabbit anti-Synaptotagmin</td>
			<td>Gift from Hugo Bellen</td>
			<td></td>
			<td>Jan-00</td>
		</tr>
		<tr>
			<td>Mouse anti-Cysteine string protein</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>DCSP-1(ab49)</td>
			<td>1/10 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit)&#160;</td>
		</tr>
		<tr>
			<td>Mouse anti-Bruchpilot</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>nc82</td>
			<td>Jan-50</td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa Fluor 488</td>
			<td>Life technologies</td>
			<td>A11029</td>
			<td>1/200</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 568</td>
			<td>Life technologies</td>
			<td>A11011</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Zenon Alexa Fluor 568 Mouse IgG1 Labeling Kit</td>
			<td>ThermoFisher</td>
			<td>Z25006</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Confocal microscope or fluorescence microscope</td>
			<td>Leica SP5</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio imager</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Mac or Pc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

two algorithms for high-throughput and multi-parametric quantification of drosophila neuromuscular junction morphology

la morfología sináptica está estrechamente relacionada con la eficacia sináptica, y en muchos casos los defectos morfológicos sinapsis en última instancia conducir a un mal funcionamiento sináptico. La unión Drosophila larval neuromuscular (NMJ), un modelo bien establecido para las sinapsis glutamatérgicas, ha sido ampliamente estudiado por décadas. La identificación de las mutaciones que causan defectos morfológicos NMJ reveló un repertorio de genes que regulan el desarrollo de sinapsis y la función. Muchos de ellos fueron identificados en estudios a gran escala que se centraron en los enfoques cualitativos para detectar anomalías morfológicas de la Drosophila NMJ. Un inconveniente de los análisis cualitativos es que muchos jugadores sutiles que contribuyen a la morfología NMJ probable que pasan desapercibidos. Mientras que se requieren análisis cuantitativos para detectar las diferencias morfológicas más sutiles, tales análisis no son todavía comúnmente realizan porque son laboriosos. Este protocolo describe en detalle dos algoritmos de análisis de imagen &quot;Drosophila </em&gt; NMJ Morfometría&quot; y &#39;Drosophila NMJ Bouton Morfometría&#39;, disponible como macros compatibles con Fiji, para el análisis morfométrico cuantitativo, precisa y objetiva de la Drosophila NMJ. Esta metodología se desarrolló para analizar terminales NMJ immunolabeled con los marcadores utilizados comúnmente Dlg-1 y . Brp Además, su aplicación más amplia a otros marcadores como Hrp, Csp y Syt se presenta en este protocolo Las macros son capaces de evaluar nueve características NMJ morfológicas:. zona NMJ, perimetrales NMJ, número de boutons, longitud NMJ, NMJ rama más larga longitud, el número de islas, número de ramas, el número de puntos de ramificación, el número de zonas activas en el terminal NMJ.

Watch this Scientific Journal Video about Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology at JoVE.com

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Se crearon dos algoritmos de análisis de imagen, &quot;Drosophila NMJ morfometría&quot; y &quot;Drosophila NMJ Bouton morfometría&quot;, para cuantificar automáticamente nueve características morfológicas de la unión neuromuscular Drosophila (NMJ).

Dos algoritmos para de alto rendimiento y multi-paramétrico Cuantificación de<em&gt; Drosophila</em&gt; La unión neuromuscular Morfología

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

two-algorithms-for-high-throughput-multi-parametric-quantification

Research

JoVE Journal

Neuroscience

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

9.6K vistas.  Radboud University Medical Center.  Se crearon dos algoritmos de análisis de imagen, "Drosophila NMJ morfometría" y "Drosophila NMJ Bouton morfometría", para cuantificar automáticamente nueve características morfológicas de la unión neuromuscular Drosophila (NMJ). 

Video: Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

La disección y la imagen de zonas activas en el Drosophila La unión neuromuscular

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Investigación

Neurociencias

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

En electroporación in vivo de la retina del ratón en desarrollo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Secuencial Foto-blanqueamiento para Precisar las células individuales de Schwann en la unión neuromuscular

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

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 ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Detección de<em&gt; In Situ</em&gt; Complejos proteína-proteína en la<em&gt; Drosophila</em&gt; Larval unión neuromuscular Usando proximidad Ligadura Ensayo

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to a certain degree, many animals have to be analyzed, to obtain statistically significant data. This in turn requires a computer assisted automated quantification of locomotion patterns. To obtain high contrast images of almost translucent and small moving objects, a novel imaging technique based on frustrated total internal reflection called FIM was developed. In this setup, animals are only illuminated with infrared light at the very specific position of contact with the underlying crawling surface. This methodology results in very high contrast images. Subsequently, these high contrast images are processed using established contour tracking algorithms. Based on this, we developed the FIMTrack software, which serves to extract a number of features needed to quantitatively describe a large variety of locomotion characteristics. During the development of this software package, we focused our efforts on an open source architecture allowing the easy addition of further modules. The program operates platform independent and is accompanied by an intuitive GUI guiding the user through data analysis. All locomotion parameter values are given in form of csv files allowing further data analyses. In addition, a Results Viewer integrated into the tracking software provides the opportunity to interactively review and adjust the output, as might be needed during stimulus integration. The power of FIM and FIMTrack is demonstrated by studying the locomotion of Drosophila larvae.

FIM is a novel, cost effective imaging system designed to track small moving objects such as C. elegans, planaria or Drosophila larvae. The accompanying FIMTrack program is designed to deliver fast and efficient data analysis. Together, these tools allow high-throughput analysis of behavioral traits.

FIM Imaging y FIMtrack: Dos nuevas vías que hagan de alto rendimiento y Análisis de Costo Efectivo Locomoción

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Ensayo de Alto Rendimiento para el Examen de Preferencias de la puesta de huevos individual<em&gt; Drosophila melanogaster</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

¿Por qué importa la cuantificación: Caracterización de fenotipos en el<em&gt; Drosophila</em&gt; Larval unión neuromuscular

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Medir la funcionalidad de la placa neuromuscular

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Cuantificación de la Drosophila Grooming Comportamiento

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...