Damos las gracias a Joachim Erber para introducirnos en el paradigma de aprendizaje táctil en las abejas melíferas. Este trabajo fue apoyado por el Cluster de Excelencia 277 CITEC, financiado en el marco de la Iniciativa de Excelencia alemán.

1. Preparación de las abejas <ol><li> Recolectoras de néctar o polen son capturados en el campo ya sea desde un alimentador de sacarosa o directamente desde la entrada de la colmena cuando regresaba de un viaje de forrajeo. Cada abeja es capturado en un vial de vidrio que se cierra con un tapón de espuma y se lleva inmediatamente al laboratorio para su posterior manipulación. </li><li> En el laboratorio, las abejas se capturaron brevemente enfrió en el refrigerador a 4 ° C hasta que muestren signos primera de inmovilidad. </li><li> Cada abeja inmovilizado está montado en un pequeño tubo de metal con cinta adhesiva entre la cabeza y el tórax y en el abdomen. Se debe tener cuidado de que la trompa y las antenas se mueven libremente. </li><li> Pintar los ojos compuestos y ocelos de la abeja fijo con pintura blanca (por ejemplo, libre de solventes Tipp-Ex) para ocluir la visión. </li><li> Agregar una pequeña gota de cera derretida detrás de la cabeza de la abeja para fijarlo a la cinta entre la cabeza y el tórax para evitar movimientos de la cabeza durante las grabaciones. </li><li> Marque cada abeja con un número en la cinta para una mejor identificación y coloque el tubo con la abeja fijo en un ambiente húmedo para evitar la deshidratación. </li><li> Alimentar cada sola abeja durante 5 segundos con gotitas de una solución de sacarosa al 30% se presentaron con una jeringa y dejar que todas las abejas recuperar durante 30 min antes de comenzar con el protocolo acondicionado táctil. </li></ol> 2. Aire Táctil <ol><li> Antes de acondicionamiento, cada abeja tiene que ser probada por la respuesta de extensión probóscide (PER) a un estímulo de sacarosa al 30% aplicado a las antenas. De esta manera la punta de la trompa tiene que cruzar una línea virtual entre las mandíbulas abiertas. Deseche todas las abejas que no responden con un PER para el estímulo de sacarosa. </li><li> Para el acondicionamiento táctil utilizar un cubo de latón (por ejemplo, 3 x 5 mm) con una superficie lisa o un patrón de grabado, por ejemplo, horizontal o ranuras verticales que forman una rejilla con 150 micras de longitud de onda, como el estímulo condicionado (CS). Fo el estímulo no condicionado (EE.UU.) utilizar una solución de sacarosa al 30% (azúcar doméstico, diluido en agua). </li><li> El cubo de latón (CS) se coloca en un soporte en un micromanipulador (por ejemplo, Märzhäuser MM33) para asegurar el posicionamiento exacto durante el procedimiento de acondicionado. Los EE.UU. se presenta a la abeja con una jeringa llena de una solución de sacarosa al 30%. </li><li> El procedimiento de acondicionamiento se compone de cinco emparejamientos de la estímulo táctil (CS) y solución de sacarosa (EE.UU.), con un intervalo entre ensayos (ITI) de 5 min. Coloque una sola abeja en frente de la micromanipulador con el estímulo táctil montada (CS). Coloque la CS lentamente, de tal manera que la superficie del estímulo táctil es paralela a la cabeza de la abeja (Figura 1 A y B). La distancia entre el animal y el objeto táctil debe estar en el rango del radio antenal de trabajo de la abeja probado, es decir, la abeja debe ser capaz de escanear el estímulo táctil en una posición cómoda wi-ésimo ambas antenas. En el ejemplo mostrado en la Figura 1, la distancia era de 3 mm. Deje que la abeja escanear el estímulo táctil (CS) durante 5 segundos. Después de la primera sec 3, presentan una gotita de la sacarosa al 30% (EE.UU.) solución con una jeringa bajo la proboscis. Utilice la punta de la jeringa para elevar suavemente la probóscide. Estimulación sacarosa bajo la probóscide provocará la incondicionado PER 15. Deje que la abeja a lamer la recompensa sacarosa. Use un cronómetro con una señal de alarma para mantener los intervalos de tiempo exactos durante el acondicionamiento. </li><li> El PER acondicionado se usa como una medida para el éxito de aprendizaje de una abeja. Después de que los emparejamientos primera recompensa, las abejas comienzan a responder a la presentación CS, por extensión, de su trompa, lo que indica que esperan que la recompensa futura. A probóscide completamente extendidas observadas en cualquier momento durante la ventana de tiempo de 3 segundos de presentación del estímulo táctil y antes de la presentación de sacarosa se obtuvo una respuesta positiva. No hay respuesta se cuenta negativa. El occurrence de la PER ha de señalar por el experimentador. El porcentaje de abejas que muestran el PER durante la presentación de CS se traza para cada ensayo. </li></ol> 3. Grabaciones cinemáticos <ol><li> Movimientos antenales de una sola abeja aprovechada se graban con una cámara de vídeo digital con un lente macro adecuada (por ejemplo Basler A602f-2 equipado con TechSpec VZM 200, funciona a 50 fps a través de una conexión de cable de fuego). La cámara se coloca por encima del animal en una vista de arriba hacia abajo (Figura 1 A). En la Figura 1, la resolución espacial de las grabaciones es 0,02 mm / pixel. </li><li> Calibrar la cámara mediante la grabación de imágenes individuales de un tablero de ajedrez 10 x 10 mm con una longitud de arista de 1 mm a partir de diferentes orientaciones en el marco del objetivo de la cámara. La calibración se puede hacer con la caja de la cámara herramienta de calibración 16 Matlab. </li><li> Coloque una sola abeja fijo por debajo del objetivo de la cámara. Presentar el estímulo táctil, fijado a un micromanipulador, Para el animal. Proceder de la misma manera como se describe para el acondicionamiento táctil, y registrar el movimiento antenal mientras que la abeja se analizó el objeto. Es importante que toda la gama de trabajo antenal y el estímulo táctil son visibles. Las opciones de los patrones de estímulos probados y números de ensayo dependerá del diseño experimental. </li></ol> 4. Análisis de Datos <ol><li> El cálculo de la imagen de fondo se realiza en Matlab. En primer lugar, el valor medio de escala de grises en el tiempo tiene que ser calculado para cada píxel. Los objetos estáticos, como la cabeza fija de la abeja y el estímulo táctil, constituirán el fondo de la imagen. Los objetos en movimiento, como las dos antenas, no será parte de los antecedentes. </li><li> Cada fotograma de la grabación de vídeo tiene que ser cargado de forma secuencial, y la diferencia entre la trama actual y la imagen de fondo tiene que ser calculado. El resultado de esta resta hace hincapié en las partes de la imagen que se están moviendo de marco a marco. Identificacióneally, las antenas de las abejas son las únicas áreas de valores distintos de cero (Figura 1 C). </li><li> Para el tratamiento posterior, las dos mayores áreas con valores de los píxeles que no son cero se supone que son las antenas. Para ambas antenas una máscara binaria se ha de generar para cada píxel que contiene un valor de 1, si el píxel pertenece a la antena y el valor 0 de lo contrario. Esta máscara sirve de base para la localización de las puntas antenales más tarde. Para obtener una máscara preliminar para toda la imagen gris del valor de cada píxel de la imagen de diferencia se compara con un umbral predefinido. Dado que nosotros nos encargamos de ruido a finales de este umbral se elige para ser bastante baja. La máscara preliminar entonces todavía lleva dos tipos de errores: En primer lugar, debido al ruido de la imagen pequeñas regiones son todavía parte de la máscara. En segundo lugar, las zonas que pertenecen a la antena podría no ser necesariamente completamente conectada. En estas últimas, se produce si el fondo tiene aproximadamente la misma luminiscencia como la antena. Para eliminar estos artefactos, estándaroperación de procesamiento de imagen morfológica se aplican, es decir, una combinación de erosión y dilatación imagen, confiando en las funciones estándar de Matlab imerode y imdilate respectivamente (ver 17 pp 158-205 para una explicación más detallada). Después de que el proceso de eliminación de ruido de las hipótesis antenas son todavía contenido dentro de la misma máscara. Por lo tanto, como paso siguiente la máscara binaria está agrupado por áreas disjuntas utilizando el estándar de Matlab función bwlabel (ver 17 pp 40-48 para más detalles sobre el algoritmo de segmentación de imágenes). </li><li> El número de píxeles por racimo se cuenta y los dos mayores grupos seleccionados. El centro de gravedad se calcula para distinguir entre la antena izquierda y derecha (Figura 1D). </li><li> La punta antenal para cada antena puede ser definido como el píxel en un grupo con el valor más alto en la dirección proximal a distal (véase la Figura 1D). </li></ol>

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Preparación de las abejas: Recogida y se fijan las abejas debe hacerse rápidamente, con el fin de mantener el nivel de estrés de la baja animal. El estrés tiene un efecto sobre la capacidad de respuesta PER-y, por lo tanto, podría tener un efecto indirecto en el rendimiento de aprendizaje en las abejas 19, 20. El nivel de estrés se puede disminuir mediante la colocación de los viales de vidrio con las abejas directamente en hielo inmediatamente después de la recolección para inmovili...

<table border="1"><tbody><tr><td> Nombre del reactivo / material </td><td> Empresa </td><td> Número de Catálogo </td><td> Comentarios </td></tr><tr><td> Recolectoras de polen, Apis mellifera L. </td><td></td><td></td><td> Urticaria privadas </td></tr><tr><td> Viales de vidrio (22 x 45 mm) </td><td> Carl Roth GmbH &amp; Co </td><td> X655.1 </td><td></td></tr><tr><td> Tubos de latón, 10 x 30 mm </td><td></td><td></td><td> De producción propia </td></tr><tr><td> Cubo de latón, 3 x 5 mm </td><td></td><td></td><td> De producción propia </td></tr><tr><td> Tipp-Ex (sin disolventes) </td><td> Tipp-Ex GmbH &amp; Co KG </td><td></td><td></td></tr><tr><td> Solución de sacarosa (30%) </td><td></td><td></td><td> Suger doméstico diluido en agua </td></tr><tr><td> Etanol (70%) </td><td> Carl Roth GmbH &amp; Co </td><td> 9065,3 &lt;/ Td&gt; <td></td></tr><tr><td> Jeringa (1 ml) y la aguja (0,6 x 25) </td><td> Carl Roth GmbH &amp; Co </td><td> 59,1 </td><td></td></tr><tr><td> Cronómetro </td><td> Carl Roth GmbH &amp; Co </td><td> L423.1 </td><td></td></tr><tr><td> Micromanipulador </td><td> Märzhäuser MM33 </td><td> 00-42-101-0000 </td><td></td></tr><tr><td> Cámara de vídeo digital </td><td> Basler A602f-2 </td><td></td><td></td></tr><tr><td> Lente macro para cámaras </td><td> TechSpec VZM 200 </td><td></td><td></td></tr><tr><td> Matlab R2009b </td><td> The MathWorks </td><td></td><td></td></tr></tbody></table>

tactile conditioning and movement analysis of antennal sampling strategies in honey bees (apis mellifera l.)

Las abejas de miel (Apis mellifera L.) son insectos eusociales y bien conocido por su compleja división del trabajo y la capacidad de aprendizaje asociativo 1, 2. Las abejas obreras pasar la primera mitad de su vida en el interior de la colmena oscura, donde se está cuidando las larvas o la construcción de la regular peines hexagonales para la alimentación (por ejemplo polen o néctar) y cría 3. Las antenas son antenas multi-sensoriales extraordinarias y desempeñan un papel fundamental en diversas tareas táctiles mediadas por 4, incluyendo la construcción de la colmena 5 y reconocimiento de patrones 6. Más tarde en la vida, cada abeja sale de la colmena para buscar comida. A continuación, una abeja tiene que aprender a discriminar fuentes rentables de alimentos, memorizar su ubicación, y la comunicará a sus compañeros de nido 7. Las abejas utilizan diferentes señales florales como colores u olores 7, 8, sino también señales táctiles de la superficie del pétalo 9 para formar memorias multisensoriales de la fuente de alimento. En virtud de laboratorio conditions, las abejas pueden ser entrenados en un paradigma de aprendizaje apetitivo para discriminar las características táctiles de objetos, tales como bordes o surcos con sus antenas 10, 11, 12, 13. Este paradigma de aprendizaje está estrechamente relacionada con el condicionamiento clásico de la respuesta olfativa de extensión proboscis (PER) en abejas enjaezados 14. La ventaja del modelo de aprendizaje táctil en el laboratorio es la posibilidad de combinar los experimentos de comportamiento en el aprendizaje con diversas mediciones fisiológicas, incluyendo el análisis del patrón de movimiento antenal.

En el siguiente experimento estudiamos cómo afecta la conducta de aprendizaje táctil muestreo antenal en las abejas melíferas. Para ello, se monitoreó el movimiento de la punta antenal en abejas naïve y acondicionado antes y durante la presentación de un estímulo táctil. En primer lugar, el movimiento espontáneo antenal de un grupo de recolectoras de polen (N = 42) fue registrada por 1 min. Una media de las abejas (N = 21) se acondicionó a continuación, por el emparejamiento de ci...

Watch this Scientific Journal Video about Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L. at JoVE.com

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

En este protocolo se muestra cómo acondicionar aprovechado las abejas a los estímulos táctiles e introducir una técnica de captura de movimiento 2D para analizar la cinemática de escala fina patrón de muestreo antenal.

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 ...

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Neuroscience

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

10.5K vistas.  Bielefeld University. En este protocolo se muestra cómo acondicionar aprovechado las abejas a los estímulos táctiles e introducir una técnica de captura de movimiento 2D para analizar la cinemática de escala fina patrón de muestreo antenal.

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

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 ...

Investigación

Neurociencias

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Automatizado de Análisis de la morfología neuronal Sholl digitalizados en múltiples escalas

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

La cultura de la motora primaria y las neuronas sensoriales en los medios de comunicación definidas en Electrospun poli-L-láctico andamios nanofibras

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

El calcio de la imagen del olor de respuestas evocadas en el Drosophila Lóbulo antenal

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Las mediciones electrofisiológicas y Análisis de la nocicepción en los bebés humanos

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Determinación rápida del umbral nociceptivo térmico en ratas diabéticas

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

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

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

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

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

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 ...

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 ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

El uso de condicionamiento de clásico de Eyeblink rastro para evaluar disfunción hipocampal en un modelo de rata de trastornos del espectro alcohólico Fetal

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Drosophila condicionamiento del corte como una medida de aprendizaje y memoria

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...