Los autores desean agradecer a Erin Fennern para ayudar experimento. Este trabajo fue financiado por el Consejo de Investigación de Noruega (180.504, 185.306 y 191.699), PEW Charitable Trust y el Instituto Nacional sobre el Envejecimiento (NIA P01 AG22500).

Parte 1: RNAi mediada desmontables de dos genes 1. dsRNA síntesis <ol><li> Diseño de cebadores dsRNAs dirigidos vg, USP y una proteína de control del gen que codifica la fluorescencia verde (GFP), que no está en la miel de abeja genoma: primers fueron diseñados utilizando software libre en línea Primer3 ( <a href="http://frodo.wi.mit.edu/" target="_blank">http://frodo.wi.mit.edu/</a> ) . </li><li> síntesis de dsRNA para VG, USP y GFP: uso RiboMax T7 sistema de producción de ARN de Promega para la transcripción in vitro. </li><li> dsRNA purificación: <ol><li> Desnaturalización y renaturalización: el calor del dsRNA a 85 ° C durante 5 minutos y dejar enfriar a temperatura ambiente durante 1 hora. </li><li> Tratamiento con DNasa I: añadir 1 l de DNasa I en cada reacción de dsRNA y mezclarlas con un movimiento rápido de los tubos. Incubar durante 15 min a 37 ° C. </li><li> Añadir 150 l de agua libre de nucleasa y 750 l TRizol - LS en cada reacción y mezclar suavemente les invErting el tubo. Se incuban las muestras durante 5 min a 30 ° C. </li><li> Añadir 200 l de cloroformo a cada muestra. Mezclar enérgicamente durante 20 segundos. Centrifugar las muestras durante 15 min a 12.000 xg a 4 ° C. </li><li> Transferir la fase sobrenadante de cada tubo nuevo y añadir 500 l de alcohol isopropílico en cada tubo. Mezclar invirtiendo el tubo. Incubar la mezcla durante 20 min a -20 ° C. Centrifugar durante 10 minutos a 12.000 xga 4 º C. </li><li> Eliminar el sobrenadante y el uso de 1,000 l de etanol 75% para lavar el sedimento. Después de centrifugar el tubo durante 5 min a 7500 xg a 4 ° C, el aire seco del dsRNA de pellets, y el uso de agua libre de nucleasa para disolver el precipitado. Con el fin de garantizar la eficacia del gen de la regulación a la baja, la concentración de ARN de doble cadena debe estar alrededor de 9 -10 g / l. </li></ol></li></ol> 2. dsRNA inyección abdominal Por gen desmontables doble, hay dos estrategias: 1) una sola inyección: dsRNA mezcla de dos genes y Inject la mezcla; 2) de dos días de inyección: inyectar la primera orientación dsRNA un gen en el primer día, e inyectar el segundo ARN de doble cadena en las mismas abejas en el segundo día. <ol><li> Chill recientemente surgió abejas en un refrigerador 4 ° C durante 1-2 min. Cuando se inmovilizan las abejas, las abejas 3-4 montar en paralelo en placas de Petri llenas de cera sólida con alfileres de insectos cruzadas entre el abdomen y thoraces. </li><li> Enfríe las abejas de nuevo en un ° C refrigerador 4. Asegúrese de que las abejas son completamente inmóvil. Se tarda unos 1-2 min. Las abejas deben buscar suelto. Si las abejas se vuelven arrugadas o retorcida, significa que se enfrían demasiado tiempo que deben evitarse. Tiempo libre demasiado provoca una alta mortalidad, pero inmovilización completa es importante ya que cualquier movimiento aumenta el tamaño de la herida y la mortalidad. </li><li> Coloque una aguja desechable 30G (BD) en un micro jeringa Hamilton. Tome 3 l dsRNA y asegúrese de que no hay burbujas de aire en la jeringa. La aguja se introduce a un lado del abdomen para evitar la damaging órganos internos. Presione el émbolo de la jeringa lentamente para permitir que el ARN de doble cadena para ser absorbido. Desde dsRNA es muy viscoso, que se necesita 2-3 seg para ser completamente expulsado de la jeringa. Tras completamente empujando hacia abajo el émbolo, deje la aguja en la herida durante 4-5 segundos. Observar las abejas para 3-5 segundos después de las inyecciones. Si un hemolinfa fugas gotita de la herida, desechar la abeja. </li><li> Usar diferentes colores para marcar sus Thoraces correspondientes a diferentes tratamientos. En este punto, si se utiliza la estrategia de inyección única, las abejas pueden colocarse de nuevo en una colonia después de 1 hora de observación. Sin embargo, si se utiliza la estrategia de inyección de dos días, después de haber sido inyectados con la primera dsRNA, las abejas deben ser colocados en una jaula de malla de cilindro con miel suministrado en el lado y se mantuvieron en una incubadora a 34 ° C y 80% de humedad . </li><li> En el segundo día, siga los pasos 2.2 y 2.3 para relajarse de las abejas en las jaulas de malla y montarlos en placas de cera. Realizar inyecciones con la segunda dsRNA como es descricama en el paso 2.4. </li><li> Que las abejas se recuperan a temperatura ambiente durante 1 hora y los introducen en una colonia. </li></ol> Nota: efectos Desmontables pueden ser detectados por RT-PCR cuantitativa (QRT-PCR) y el Western Blot. En general, la eficacia de abatimiento varía y depende de muchos factores. En nuestro estudio, la caída de dos genes puede ser detectada 7 días después de las inyecciones individuales y las inyecciones de dos días. Parte 2: Ensayo de respuesta de extensión probóscide (PER) 1. Recopilación de las abejas de la colmena El día 6 después de las inyecciones, recoger las abejas tratadas de la colonia sin el uso de un fumador. Recoge las abejas en las jaulas de los cilindros de malla metálica. 3 Mantenga las abejas en cada jaula. 2. Abejas de montaje para PER Enfríe las abejas a 4 ° C hasta que estén completamente inmovilizados. Monte cada abeja verticalmente en un tubo de plástico especialmente diseñada para PER. Mantenga el abdomen dentro del tubo por using dos pequeños trozos de cinta, y mantener la cabeza que sobresale del tubo. Colocar los tubos en un bastidor con dos filas de pequeñas columnas de plástico, y asignar a cada tubo (abeja) con un número de identificación. Luego ponga las abejas tratadas en los bastidores en una incubadora (34 ° C y 80% de humedad) y mantenerlos en la incubadora durante 2 horas. 3. Preparar una serie de sacarosa Water Solutions Preparar 0%, 0,1%, 0,3%, 1%, 3%, 10% y 30% de sacarosa soluciones de agua y la carga de las soluciones en jeringas con agujas G 15-20. 4. Por ensayo Toque antena de la abeja con una gotita de solución de 0% (agua), seguido de 0,1%, 0,3%, 1%, 3%, 10% y 30% de agua soluciones de sacarosa. Pruebe todas las abejas con agua primero y deje por lo menos dos minutos, ya que el intervalo entre cada solución se utiliza. Por lo tanto, solemos tener al menos 20 abejas de cada ensayo. Si una abeja se extiende plenamente sus probóscide, se cuenta una respuesta positiva. 5.Gestión de datos Sume el total de respuestas positivas para cada individuo de la puntuación respuesta gustativa (GRS). Realizar un análisis estadístico adecuado de los datos. Parte 3. Validación derribo Gene Cuerpos gordos son disecados de otro conjunto de 7-días abejas tratados antiguos. Un procedimiento TRizol estándar se utiliza para la extracción de ARN, que es seguido por tratamiento con DNasa I 24. Vg y la expresión génica USP es analizada por un paso de dos QRT-PCR. Actina se usa como un gen de referencia, ya que tiene expresiones estables en diferentes tejidos de las abejas melíferas. Publicado cebadores de PCR en tiempo real para VG y usp gen se utilizan en el experimento 24. Los datos se analizaron mediante el uso de Delta-Delta método CT 25.

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We have nothing to disclose.

Nuestro estudio es el primer intento de tumbar al mismo tiempo dos genes en los insectos adultos. Nuestros resultados muestran que las dos estrategias de dsRNA inyecciones (inyección única y de inyección de dos días) son eficaces para la supresión génica doble, simultánea y silenciamiento de los genes de VG y la USP pueden ser probados 6 días después de las inyecciones de dsRNA en abejas 8,9. Sin embargo, el gen de la eficacia caída varía en las diferentes especies de insectos seg...

Interferencia de ARN (ARNi) es basado en post-transcripcional silenciamiento génico de ARN, que se produce en una amplia variedad de organismos eucariotas. El proceso de ARNi se desencadena por precursores de ARN de doble cadena endógenos o exógenos (dsRNA). El ARN de doble cadena activa la proteína Dicer ribonucleasa que se une y corta el dsRNA a pequeños fragmentos (20-25 pb). A continuación, los pequeños fragmentos de la guía de dsRNA un reconocimiento y la escisión de los ARNm complementarios por las proteínas Argonaute, un componente de catalizador de complejo de silenciamiento inducido por ARN (RISC) 1. En los mamíferos, ARN de doble cadena de más de 30 nt, activan una respuesta antiviral (respuesta de interferón, IFN) que conduce a la degradación no específica de transcripciones de ARN 2. Sin embargo, ARN de doble cadena larga han demostrado ser eficaces y específicos en los insectos ya que existe una falta de este IFN 3. DsRNAs han sido utilizados para la regulación a la baja de los genes diana de diferentes especies de insectos. Las abejas son uno de los pionorganismos de insectos eer en el que las funciones de muchos genes importantes en el desarrollo y el comportamiento se han puesto de manifiesto mediante el uso de ARN de doble cadena 4,5. Varios métodos de entrega dsRNA se han realizado en las abejas melíferas: dsRNA alimentación regula a la baja de manera eficiente expresión del gen diana en la miel larvas de abeja 6, mientras que la inyección de dsRNA es un método eficaz para la caída de genes en embriones de abejas 4 y abejas adultas 7,8. Efectos de derribo Gene exhibidos por la aplicación de dsRNA a los insectos son transitorios y localizados. Los estudios han demostrado que ambas inyecciones dsRNA abdominales y torácicos inyecciones dsRNA suprimen eficazmente la expresión de genes diana en las células abdominales de grasa corporal de los insectos 9,10. DsRNA se inyecta en las cavidades abdominales y torácicas y las células de grasa corporal son capaces de asumir el dsRNA de la hemolinfa donde las células están bañadas 10. Sin embargo, los genes en otros órganos, como los ovarios y el cerebro, no pueden ser dirigidos por cualquierainyecciones abdominales o torácicas. Con el fin de orientar los genes en la miel de abeja cerebro, también se ha realizado la inyección de dsRNA cerebro, lo que influye efectivamente expresión del gen diana en las zonas locales del cerebro 11. Aquí, sólo documentamos dsRNA inyección abdominal que es más comúnmente utilizado en las abejas adultas. ARNi ha sido utilizado principalmente para llegar a un solo gen y ha sido una herramienta poderosa para revelar la función del gen. Sin embargo, cualquier gen no está aislado de los demás, es en redes reguladoras complejas. Una clave para entender un proceso biológico es diseccionar cómo los genes interactúan entre sí, lo que requiere manipulaciones simultáneas de múltiples genes en lugar de un único gen desmontables. En líneas celulares de mamíferos, los científicos han logrado inhibir simultáneamente dos o tres genes mediante el uso de sistemas de suministro de 12 o multi-microRNA (miRNA) diseños horquilla 13. Pero en los insectos, múltiples caídas de genes todavía no probado. En este sentido, Presenestrategias de inyección diferentes t que puede alcanzar una caída de dos genes. Nos dirigimos a dos genes: vitelogenina (VG) que codifica un precursor de proteína de yema de huevo, y ultraspiracle (USP) que codifica un receptor putativo de la hormona juvenil (JH) y puede servir como un factor de transcripción mediación de las respuestas a JH 14 en las abejas de miel. Vg y JH regulan entre sí en un circuito de retroalimentación 15 y están involucrados en la miel de abeja comportamiento regla 9. Con el derribo de dos genes, nos perturban tanto Vg y las vías de JH, y descubrir cómo afectan conjuntamente comportamiento de las abejas de miel y la fisiología y cómo vg, USP y JH interactúan 9. Percepción gustativa es un predictor del comportamiento de la miel de abeja comportamiento social 16. En términos de desarrollo de comportamiento, las abejas nido con alta percepción gustativa conducta madura rápido, y por lo general forraje temprano en la vida y prefieren recoger el polen 16,17. Aunque m reglamentarioechanisms subyacen a la percepción gustativa aún no están claros, los estudios han demostrado que la percepción gustativa está relacionada con metabolismos energéticos internos 9, la secreción hormonal 18,19 y biogenética amina dirigen a las vías 20. Tanto Vg y JH son importantes reguladores hormonales modulación de la percepción gustativa 7,21. En el laboratorio, una variación de la percepción gustativa en las abejas de miel puede ser evaluada probando la respuesta de extensión proboscis (PER) a diferentes soluciones de sacarosa. Cada abeja se prueba tocando tanto sus antenas con una gotita de agua, seguido por una serie ascendente concentración de 0,1, 0,3, 1, 3, 10, 30% de sacarosa. Una respuesta positiva se observó si una abeja se extiende completamente sus trompa cuando una gota de agua o sacarosa se toca para cada antena. Basado en el número de respuestas positivas a las soluciones, el nivel de la percepción gustativa de cada individuo se puede determinar 16. Sin embargo, la aplicación del POR no está limitada a la medición de gpercepción ustatory. El PER es también un método eficaz para poner a prueba el estado metabólico de las abejas, tales como el hambre saciedad vs. Las abejas con una mayor respuesta a la sacarosa tienen más hambre en general (Wang y Amdam, datos no publicados). Por otra parte, el paradigma POR también se puede utilizar en el aprendizaje y la memoria asociativa en las abejas de miel. En este caso las abejas serán entrenados para asociar la presencia de agua de sacarosa con un olor. Cuando las abejas aprenden la asociación, sólo la presencia del olor puede evocar una respuesta positiva trompa sin recompensar con la sacarosa 22,23. En este vídeo, se muestra cómo realizar PER para evaluar la percepción gustativa que se ha conectado con vg y usp doble caída en un estudio anterior 9.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
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			<td>GE Healthcare PCR beads in 96 well plate</td>
			<td>GE Healthcare</td>
			<td>27-9557-01</td>
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			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
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			<td>RiboMax T7 RNA production system</td>
			<td>Promega</td>
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			<td>Chloroform:Isoamyl alcohol 24:1</td>
			<td>Sigma-Aldrich</td>
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			<td>isopropyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>I9516</td>
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			<td>Hamilton micro syringe</td>
			<td>Hamilton</td>
			<td>80301</td>
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			<td>30G BD disposable needles</td>
			<td>BD Biosciences</td>
			<td>305106</td>
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			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1 ml Syringe</td>
			<td>BD Biosciences</td>
			<td>30960</td>
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		 </tr>
		 <tr>
			<td>Stereo dissection microscope</td>
			<td>Leica Microsystems</td>
			<td>S6D</td>
			<td></td>
		 </tr>
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			<td>Insect pins</td>
			<td>Fine Science Tools</td>
			<td>26000-25</td>
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			<td>Wax plate</td>
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		</tbody>
 </table>

rnai-mediated double gene knockdown and gustatory perception measurement in honey bees (apis mellifera)

Este vídeo muestra novedosas técnicas de interferencia de ARN (RNAi), que regulan negativamente dos genes simultáneamente en las abejas que utilizan inyecciones de ARN de doble cadena (dsRNA). También se presenta un protocolo de respuesta de extensión proboscis (POR) de ensayo para la medición de la percepción gustativa. RNAi mediada desmontables gen es una técnica eficaz disminuyendo la expresión del gen diana. Esta técnica se utiliza generalmente para la sola manipulación genética, pero tiene limitaciones para detectar las interacciones y los efectos conjuntos entre genes. En la primera parte de este video, se presentan dos estrategias para tumbar al mismo tiempo dos genes (llamados genes desmontables dobles). Mostramos ambas estrategias son capaces de suprimir de forma eficaz dos genes, vitelogenina (VG) y ultraspiracle (USP), que están en un bucle de retroalimentación reguladora. Este enfoque caída doble gen se puede utilizar para diseccionar interrelaciones entre los genes y se puede aplicar fácilmente endiferentes especies de insectos. La segunda parte de este video es una muestra de ensayo de respuesta de extensión probóscide (PER) en las abejas de miel después de que el tratamiento de la caída de dos genes. El ensayo PER es una prueba estándar para medir la percepción gustativa de las abejas, que es un indicador clave de la rapidez con la maduración del comportamiento de una abeja es. Mayor percepción gustativa de las abejas nido indica el creciente desarrollo de la conducta que a menudo se asocia con una edad más temprana de inicio de la búsqueda de alimento y forraje especialización en el polen. Además, por ensayo se puede aplicar para identificar estados metabólicos de saciedad o hambre en las abejas de miel. Por último, por ensayo en combinación con el emparejamiento diferentes estímulos de olor para el acondicionamiento de las abejas también es ampliamente utilizado para el aprendizaje y la memoria en los estudios de las abejas de miel.

Tanto los dos días de la inyección estrategias de inyección única y redujeron significativamente vg (One-way ANOVA, p &lt;0.001) (Figura 2A) y USP (One-way ANOVA, p &lt;0.001) (Figura 2B), los niveles de transcripción en las abejas de miel seis días después de la dsRNA se inyectó. La supresión de la USP transcripción mediante el uso de la inyección única con la mezcla de ARN de doble cadena y la inyección de dos días con VG primera y seg...

Watch this Scientific Journal Video about RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera at JoVE.com

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

En este protocolo, se describen dos estrategias que simultáneamente suprimen dos genes (genes desmontables doble) en las abejas de miel. A continuación te presentamos cómo utilizar la respuesta de extensión probóscide (PER) de ensayo para estudiar el efecto de la caída de dos genes de la miel de abeja percepción gustativa.

RNAi mediada Doble derribo Gene y medición percepción gustativa de las abejas (<em&gt; Apis mellifera</em&gt;)

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

rnai-mediated-double-gene-knockdown-gustatory-perception-measurement

Research

JoVE Journal

Neuroscience

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

14.8K vistas.  Arizona State University. En este protocolo, se describen dos estrategias que simultáneamente suprimen dos genes (genes desmontables doble) en las abejas de miel. A continuación te presentamos cómo utilizar la respuesta de extensión probóscide (PER) de ensayo para estudiar el efecto de la caída de dos genes de la miel de abeja percepción gustativa.

Video: RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Doble fluorescencia In situ La hibridación en las secciones del cerebro fresco

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Investigación

Neurociencias

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

Farmacología del Comportamiento en el condicionamiento clásico de la respuesta de Extensión probóscide en abejas de la miel ( Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

Creating Objects and Object Categories for Studying Perception and Perceptual Learning

In order to quantitatively study object perception, be it perception by biological systems or by machines, one needs to create objects and object categories with precisely definable, preferably naturalistic, properties1. Furthermore, for studies on perceptual learning, it is useful to create novel objects and object categories (or object classes) with such properties2.
Many innovative and useful methods currently exist for creating novel objects and object categories3-6 (also see refs. 7,8). However, generally speaking, the existing methods have three broad types of shortcomings.
First, shape variations are generally imposed by the experimenter5,9,10, and may therefore be different from the variability in natural categories, and optimized for a particular recognition algorithm. It would be desirable to have the variations arise independently of the externally imposed constraints.
Second, the existing methods have difficulty capturing the shape complexity of natural objects11-13. If the goal is to study natural object perception, it is desirable for objects and object categories to be naturalistic, so as to avoid possible confounds and special cases.
Third, it is generally hard to quantitatively measure the available information in the stimuli created by conventional methods. It would be desirable to create objects and object categories where the available information can be precisely measured and, where necessary, systematically manipulated (or 'tuned'). This allows one to formulate the underlying object recognition tasks in quantitative terms.
Here we describe a set of algorithms, or methods, that meet all three of the above criteria. Virtual morphogenesis (VM) creates novel, naturalistic virtual 3-D objects called 'digital embryos' by simulating the biological process of embryogenesis14. Virtual phylogenesis (VP) creates novel, naturalistic object categories by simulating the evolutionary process of natural selection9,12,13. Objects and object categories created by these simulations can be further manipulated by various morphing methods to generate systematic variations of shape characteristics15,16. The VP and morphing methods can also be applied, in principle, to novel virtual objects other than digital embryos, or to virtual versions of real-world objects9,13. Virtual objects created in this fashion can be rendered as visual images using a conventional graphical toolkit, with desired manipulations of surface texture, illumination, size, viewpoint and background. The virtual objects can also be 'printed' as haptic objects using a conventional 3-D prototyper.
We also describe some implementations of these computational algorithms to help illustrate the potential utility of the algorithms. It is important to distinguish the algorithms from their implementations. The implementations are demonstrations offered solely as a 'proof of principle' of the underlying algorithms. It is important to note that, in general, an implementation of a computational algorithm often has limitations that the algorithm itself does not have.
Together, these methods represent a set of powerful and flexible tools for studying object recognition and perceptual learning by biological and computational systems alike. With appropriate extensions, these methods may also prove useful in the study of morphogenesis and phylogenesis.

We describe a novel methodology for creating naturalistic 3-D objects and object categories with precisely defined feature variations. We use simulations of the biological processes of morphogenesis and phylogenesis to create novel, naturalistic virtual 3-D objects and object categories that can then be rendered as visual images or haptic objects.

Creación de objetos y categorías de objetos para el estudio de la percepción y el aprendizaje perceptivo

In order to quantitatively study object perception, be it perception by biological systems or by machines, one needs to create objects and object ...

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

Utilizando asequible Estructuras de LED para fotos-estimulación de las neuronas

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Transducción viral selectiva de adultos nacidos en las neuronas olfativas de las enfermedades crónicas En vivo Estimulación Optogenetic

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. Humans can do path integration based exclusively on visual 2-3, auditory 4, or inertial cues 5. However, with multiple cues present, inertial cues - particularly kinaesthetic - seem to dominate 6-7. In the absence of vision, humans tend to overestimate short distances (&lt;5 m) and turning angles (&lt;30&deg;), but underestimate longer ones 5. Movement through physical space therefore does not seem to be accurately represented by the brain.
Extensive work has been done on evaluating path integration in the horizontal plane, but little is known about vertical movement (see 3 for virtual movement from vision alone). One reason for this is that traditional motion simulators have a small range of motion restricted mainly to the horizontal plane. Here we take advantage of a motion simulator 8-9 with a large range of motion to assess whether path integration is similar between horizontal and vertical planes. The relative contributions of inertial and visual cues for path navigation were also assessed.
16 observers sat upright in a seat mounted to the flange of a modified KUKA anthropomorphic robot arm. Sensory information was manipulated by providing visual (optic flow, limited lifetime star field), vestibular-kinaesthetic (passive self motion with eyes closed), or visual and vestibular-kinaesthetic motion cues. Movement trajectories in the horizontal, sagittal and frontal planes consisted of two segment lengths (1st: 0.4 m, 2nd: 1 m; &plusmn;0.24 m/s2 peak acceleration). The angle of the two segments was either 45&deg; or 90&deg;. Observers pointed back to their origin by moving an arrow that was superimposed on an avatar presented on the screen.
Observers were more likely to underestimate angle size for movement in the horizontal plane compared to the vertical planes. In the frontal plane observers were more likely to overestimate angle size while there was no such bias in the sagittal plane. Finally, observers responded slower when answering based on vestibular-kinaesthetic information alone. Human path integration based on vestibular-kinaesthetic information alone thus takes longer than when visual information is present. That pointing is consistent with underestimating and overestimating the angle one has moved through in the horizontal and vertical planes respectively, suggests that the neural representation of self-motion through space is non-symmetrical which may relate to the fact that humans experience movement mostly within the horizontal plane.

An efficient way to gain insight into how humans navigate themselves in three dimensions is described. The method takes advantage of a motion simulator capable of moving observers in ways unattainable by traditional simulators. Results confirm that movement in the horizontal plane is underestimated, while vertical movement is overestimated.

MPI CyberMotion Simulator: Implementación de un simulador de movimiento Novela encargado de investigar la integración multisensorial ruta en tres dimensiones

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Aire táctil y Análisis de Movimiento de estrategias de muestreo antenales de las abejas (<em&gt; Apis mellifera</em&gt; L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

Los microelectrodos de dos cañones y concéntricos para la medición de extracelular Ion señales en el tejido cerebral

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

A Novel Behavioral Ensayo para investigar las respuestas gustativas de individual, libre de movimiento abejorros (<em&gt; Bombus terrestris</em&gt;)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

La manipulación de neurofarmacológico restringida y de vuelo libre abejas de la miel,<em&gt; Apis mellifera</em

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...