Nous tenons à remercier Joachim Erber pour nous introduire dans le paradigme de l&#39;apprentissage tactile chez les abeilles. Ce travail a été soutenu par le Pôle d&#39;excellence de 277 CITEC, financé dans le cadre de l&#39;Initiative d&#39;excellence allemande.

1. Préparation des abeilles <ol><li> Butineuses de nectar ou de pollen sont pris dans le domaine soit à partir d&#39;un chargeur de saccharose ou directement à partir de l&#39;entrée de la ruche, alors qu&#39;il rentrait d&#39;un voyage de recherche de nourriture. Chaque abeille est capturée dans un flacon en verre qui est fermé par un bouchon de mousse et pris immédiatement dans le laboratoire pour une manipulation ultérieure. </li><li> Dans le laboratoire, les abeilles capturées sont brièvement refroidi au réfrigérateur à 4 ° C jusqu&#39;à ce qu&#39;ils montrent les premiers signes de l&#39;immobilité. </li><li> Chaque abeille immobilisée est monté dans un petit tube de métal avec du ruban adhésif entre la tête et le thorax et l&#39;abdomen. Des précautions doivent être prises pour que la trompe et les antennes peuvent se déplacer librement. </li><li> Peindre les yeux composés et des ocelles de l&#39;abeille fixe avec de la peinture blanche (par exemple, sans solvant, Tipp-Ex) pour occlure vision. </li><li> Ajouter une petite goutte de cire fondue derrière la tête de l&#39;abeille pour le fixer à la bande entre la tête et le thorax pour éviter les mouvements de la tête lors des enregistrements. </li><li> Marquez chaque abeille avec un certain nombre sur la bande pour une meilleure identification et placer le tube avec l&#39;abeille fixe dans une atmosphère humide pour éviter la déshydratation. </li><li> Nourrir chaque abeille pendant 5 sec avec des gouttelettes d&#39;une solution de saccharose à 30% présentaient une seringue et laisser toutes les abeilles de récupérer pendant 30 min avant de commencer avec le protocole de conditionnement tactile. </li></ol> 2. Conditionnement tactile <ol><li> Avant le conditionnement, chaque abeille doit être testé pour la réponse extension du proboscis (PER) à un stimulus de saccharose à 30% appliqué aux antennes. Ainsi le bout de la trompe doit traverser une ligne virtuelle entre les mandibules ouvertes. Jeter toutes les abeilles qui ne répondent pas à un PER à la relance du saccharose. </li><li> Pour le conditionnement tactile utiliser un cube en laiton (par exemple 3 x 5 mm) avec une surface lisse ou un motif gravé, par exemple, horizontale ou rainures verticales formant une grille de 150 um de longueur d&#39;onde, comme le stimulus conditionné (CS). Faou le stimulus inconditionnel (États-Unis) utilisent une solution de saccharose à 30% (sucre de ménage, dilué dans de l&#39;eau). </li><li> Le cube en laiton (SM) est placé dans un support sur ​​un micromanipulateur (par exemple, Märzhäuser MM33) afin d&#39;assurer un positionnement précis pendant la procédure de conditionnement. Aux Etats-Unis est présenté à l&#39;abeille avec une seringue remplie d&#39;une solution de saccharose à 30%. </li><li> La procédure de conditionnement se compose de cinq paires de stimulus tactile (CS) et une solution de saccharose (États-Unis), avec un intervalle inter-essai (IIT) de 5 min. Placez une abeille devant le micromanipulateur avec le stimulus tactile monté (CS). Placez le CS lentement, de sorte que la surface de la stimulation tactile est parallèle à la tête de l&#39;abeille (figure 1 A et B). La distance entre l&#39;animal et l&#39;objet tactile devrait être de l&#39;ordre du rayon antennaire de travail de l&#39;abeille testée, à savoir l&#39;abeille doit être capable d&#39;analyser le stimulus tactile dans une position confortable wvec les deux antennes. Dans l&#39;exemple illustré à la figure 1, la distance était de 3 mm. Laissez l&#39;abeille analyser le stimulus tactile (CS) pendant 5 sec. Après la première section 3, présenter une gouttelette du saccharose 30% (US) avec une solution de la seringue sous la trompe. Utilisez le bout de la seringue pour soulevez doucement la trompe. La stimulation du saccharose dans la trompe suscitera l&#39;inconditionné PAR 15. Laissez l&#39;abeille à lécher la récompense du saccharose. Utiliser un chronomètre avec un signal d&#39;alarme pour maintenir des intervalles de temps précis au cours du conditionnement. </li><li> Le PER conditionné est utilisé comme mesure pour la réussite de l&#39;apprentissage d&#39;une abeille. Après les appariements premier récompensés, les abeilles commencent à réagir à la présentation CS, par extension, de leur trompe, ce qui indique qu&#39;ils attendent la récompense à venir. A trompe complètement étendus observés à tout moment pendant la fenêtre 3 sec Temps de présentation des stimuli tactiles et avant la présentation du saccharose est marqué comme une réponse positive. Aucune réponse négative est compté. Le occurrence du PER est à noter par l&#39;expérimentateur. Le pourcentage des abeilles montrant le PER lors de la présentation CS est tracée pour chaque procès. </li></ol> 3. Les enregistrements cinématiques <ol><li> Mouvements antennaires d&#39;une seule abeille exploitées sont enregistrées avec une caméra vidéo numérique avec une lentille macro approprié (par exemple Basler A602f-2 équipé d&#39;TechSpec VZM 200, fonctionnant à 50 images par seconde via une connexion fire wire). La caméra est positionnée au-dessus de l&#39;animal dans une vue de haut en bas (Figure 1). Dans la figure 1, la résolution spatiale des enregistrements est de 0,02 mm / pixel. </li><li> Calibrer l&#39;appareil par l&#39;enregistrement d&#39;images individuelles d&#39;un damier de 10 x 10 mm avec une longueur d&#39;arête de 1 mm à partir des orientations différentes dans le cadre du but caméra. Le calibrage peut être fait avec la boîte à outils Matlab appareil de calibrage 16. </li><li> Placez une seule abeille fixe en dessous de la lentille de la caméra. Présenter le stimulus tactile, fixé à un micromanipulateur, À l&#39;animal. Procéder de la même manière que décrit pour le conditionnement tactile, et d&#39;enregistrer le mouvement antennaire tandis que l&#39;abeille est la numérisation de l&#39;objet. Il est important que la gamme complète antennaire de travail et le stimulus tactile sont visibles. Les choix de configurations de stimulus testé et numéros d&#39;essai dépendent de la conception expérimentale. </li></ol> 4. Analyse des données <ol><li> Le calcul du fond de l&#39;image se fait en Matlab. Tout d&#39;abord, la valeur médiane niveaux de gris au fil du temps doit être calculé pour chaque pixel. Les objets statiques, comme la tête fixe de l&#39;abeille et le stimulus tactile, constituera le fond de l&#39;image. Les objets en mouvement, comme les deux antennes, ne fera pas partie de l&#39;arrière-plan. </li><li> Chaque trame de la vidéo enregistrée doit être chargé de manière séquentielle, et la différence entre l&#39;image courante et l&#39;image de fond doit être calculée. Le résultat de cette soustraction souligne les parties de l&#39;image qui sont en mouvement de trame en trame. Ideally, les antennes de l&#39;abeille sont les seuls domaines de valeurs non nulles (figure 1C). </li><li> Pour un traitement ultérieur, les deux plus grandes zones avec des valeurs de pixels non nuls sont supposés être des antennes. Pour les deux antennes d&#39;un masque binaire doit être généré pour chaque pixel contenant une valeur 1, si le pixel appartient à l&#39;antenne et 0 sinon valeur. Ce masque sert de base pour localiser les conseils des antennes plus tard. Pour obtenir un masque préliminaire pour l&#39;image entière de la valeur de gris de chaque pixel de l&#39;image de différence est comparée à un seuil prédéfini. Depuis nous nous occupons de bruit tard ce seuil est choisi pour être assez faible. Le masque préliminaire puis porte toujours deux types d&#39;erreurs: Premièrement, en raison de bruit de l&#39;image régions petits font encore partie du masque. Deuxièmement, les zones qui appartiennent à l&#39;antenne ne sont pas forcément être totalement connecté. Celle-ci survient surtout si le fond est à peu près la même luminescence que l&#39;antenne. Pour éliminer ces artefacts, standardopération de traitement d&#39;image morphologique sont appliquées, c&#39;est à dire une combinaison de l&#39;érosion et la dilatation d&#39;image, en s&#39;appuyant sur ​​les fonctions standard Matlab imerode et imdilate respectivement (voir 17 pp 158-205 pour plus d&#39;explications). Après le processus de débruitage des hypothèses antennes sont encore contenus dans le même masque. Par conséquent, comme prochaine étape du masque binaire est concentrée dans les zones disjointes utilisant la norme Matlab fonction bwlabel (voir 17 pp 40-48 pour plus de détails sur l&#39;algorithme de segmentation d&#39;image). </li><li> Le nombre de pixels par grappe est compté et les deux plus grandes grappes sont sélectionnées. Le centre de gravité est calculée de distinguer entre l&#39;antenne gauche et droit (figure 1D). </li><li> La pointe antennaire pour chaque antenne peut être définie comme étant le pixel dans un groupe avec la valeur la plus élevée dans la direction proximale-distale pour-(voir la figure 1D). </li></ol>

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Neurosci. 4, 46 (2010).</li><li>Scheiner, R., Barnert, M., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+water+and+sucrose+responsiveness+during+the+foraging+season+affects+proboscis+extension+learning+in+honey+bees.">Variation in water and sucrose responsiveness during the foraging season affects proboscis extension learning in honey bees.</a> Apidologie. 34, 67-72 (2003).</li><li>Mujagic, S., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sucrose+acceptance,+discrimination+and+proboscis+responses+of+honey+bees+(Apis+mellifera+L.)+in+the+field+and+the+laboratory.">Sucrose acceptance, discrimination and proboscis responses of honey bees (Apis mellifera L.) in the field and the laboratory.</a> J. Comp. Physiol. 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Préparation des abeilles: Collecte et fixant les abeilles doit être fait rapidement, afin de maintenir le niveau de stress de la dépression animal. Le stress a un effet sur ​​la réactivité PER-et, par conséquent, pourrait avoir un effet indirect sur ​​les performances d&#39;apprentissage des abeilles 19, 20. Le niveau de stress peut être diminué en plaçant les flacons de verre avec les abeilles directement sur la glace immédiatement après le prélèvement pour les immobilis...

<table border="1"><tbody><tr><td> Nom de réactif / Matériel </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Butineuses de pollen, Apis mellifera L. </td><td></td><td></td><td> Urticaire privés </td></tr><tr><td> Flacons en verre (22 x 45 mm) </td><td> Carl Roth GmbH &amp; Co </td><td> X655.1 </td><td></td></tr><tr><td> Tubes en laiton, 10 x 30 mm </td><td></td><td></td><td> Autoproduits </td></tr><tr><td> Cube en laiton, 3 x 5 mm </td><td></td><td></td><td> Autoproduits </td></tr><tr><td> Tipp-Ex (sans solvant) </td><td> Tipp-Ex GmbH &amp; Co KG </td><td></td><td></td></tr><tr><td> Solution de saccharose (30%) </td><td></td><td></td><td> Suger ménager dilué dans de l&#39;eau </td></tr><tr><td> L&#39;éthanol (70%) </td><td> Carl Roth GmbH &amp; Co </td><td> 9065,3 &lt;/ Td&gt; <td></td></tr><tr><td> Seringue (1 ml) et une aiguille (0,6 x 25) </td><td> Carl Roth GmbH &amp; Co </td><td> 59,1 </td><td></td></tr><tr><td> Stop Watch </td><td> Carl Roth GmbH &amp; Co </td><td> L423.1 </td><td></td></tr><tr><td> Micromanipulateur </td><td> Märzhäuser MM33 </td><td> 00-42-101-0000 </td><td></td></tr><tr><td> Caméra vidéo numérique </td><td> Basler A602f-2 </td><td></td><td></td></tr><tr><td> Objectif macro pour appareil photo </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.)

Les abeilles (Apis mellifera L.) sont insectes sociaux et bien connus pour leur division complexe du travail et de la capacité d&#39;apprentissage associatif 1, 2. Les abeilles ouvrières passer la première moitié de leur vie à l&#39;intérieur de la ruche obscure, où ils sont soigner les larves ou le renforcement des peignes régulière hexagonale pour la nourriture (pollen ou de nectar par exemple) et du couvain 3. Les antennes sont extraordinaires palpeurs multisensorielles et jouent un rôle crucial dans divers tactiles tâches induites par 4, y compris bâtiment de 5 et 6 ruche reconnaissance des formes. Plus tard dans la vie, chaque abeille quitte la ruche à la recherche de nourriture. Puis, une abeille doit apprendre à distinguer les sources alimentaires rentables, mémoriser leur emplacement, et les communiquer à ses compagnons de nidification 7. Les abeilles utilisent différents signaux floraux comme les couleurs ou les odeurs 7, 8, mais aussi les indices tactiles de la surface des pétales de 9 à former des souvenirs multisensoriels de la source de nourriture. En vertu de coopération entre laboratoiresnditions, les abeilles peuvent être formés dans un paradigme d&#39;apprentissage appétitive de discriminer les caractéristiques des objets tactiles, tels que des bords ou des rainures avec leurs antennes 10, 11, 12, 13. Ce paradigme d&#39;apprentissage est étroitement lié au conditionnement classique de la réponse olfactive extension du proboscis (PER) dans les abeilles harnachés 14. L&#39;avantage du paradigme de l&#39;apprentissage tactile en laboratoire est la possibilité de combiner des expériences comportementales sur l&#39;apprentissage de diverses mesures physiologiques, y compris l&#39;analyse de la forme du mouvement antennaire.

Dans l&#39;expérience suivante, nous avons étudié la façon dont l&#39;apprentissage tactile affecte le comportement d&#39;échantillonnage antennaire chez les abeilles. Pour cela, nous avons suivi le mouvement de la pointe antennaire chez les abeilles naïfs et conditionné avant et pendant la présentation d&#39;un stimulus tactile. Tout d&#39;abord, le mouvement spontané antennaire d&#39;un groupe de butineuses de pollen (N = 42) a été enregistré pendant 1 min. La moitié des abeil...

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

Dans ce protocole, nous montrons comment les abeilles état attelé à des stimuli tactiles et mettre en place une technique de capture de mouvement 2D pour analyser la cinématique de fin échelle modèle d&#39;échantillonnage antennaire.

Conditionnement tactile et d&#39;analyse des mouvements des stratégies d&#39;échantillonnage antennaires chez les abeilles (<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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Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

10.5K vues.  Bielefeld University. Dans ce protocole, nous montrons comment les abeilles état attelé à des stimuli tactiles et mettre en place une technique de capture de mouvement 2D pour analyser la cinématique de fin échelle modèle d'échantillonnage antennaire.

Vidéo: 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.

Pharmacologie comportementale dans le conditionnement classique de la réponse Extension Proboscis chez les abeilles ( Apis mellifera)

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

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Neurosciences

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.

Analyse automatisée des Sholl morphologie neuronale numérisés à de multiples échelles

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 culture de moteur primaire et neurones sensoriels de médias définis sur les échafaudages électrofilées poly-L-lactide nanofibres

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.

Imagerie de calcium des odeurs évoquées réponses dans le Drosophila Lobe antennaire

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.

Mesures électrophysiologiques et d&#39;analyse de la nociception chez les nourrissons humains

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.

Détermination rapide du seuil thermique nociceptive chez les rats diabétiques

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.

ARNi médiée double inactivation génique et de mesure de la perception gustative chez les abeilles (<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.

Manipulation neuropharmacologiques de Restrained et vol libre abeilles,<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.

L’utilisation de Trace Eyeblink conditionnement de classique pour évaluer le dysfonctionnement hippocampique dans un modèle de Rat de 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 ...

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 Courtship Conditionnement en tant que mesure de l&#39;apprentissage et de la mémoire

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