Ce travail est financé par la DFG.

Préparation d&#39;abeille et de remblayage <ol><li> Catch abeilles butineuses à la ruche et le froid sur la glace pour l&#39;immobilisation. </li><li> Mont d&#39;enregistrement en plexiglas chambres 3. Fixer les yeux et le thorax pour les murs de la chambre d&#39;enregistrement avec de la cire à point de fusion bas dur. </li><li> Tirez capillaires en verre que couramment utilisées pour l&#39;électrophysiologie et de pause à la pointe pour obtenir un diamètre de l&#39;extrémité de ca. 10 microns. Couvrir la pointe du capillaire avec de la pâte de teinture. Pâte colorante se compose d&#39;un mélange 10:01 de Fura-2 dextrane et lysine fixable tétraméthylrhodamine dextrane. </li><li> Pour la procédure de coloration immobiliser antennes avec minutie ou n-eicosane. Supprimer un morceau de la cuticule au-dessus du cerveau et des glandes et de la trachée pousser sur le côté pour permettre l&#39;accès au corps des champignons. </li><li> Injecter capillaires soit dans le soma ou région axonales des neurones du corps de champignons. Cela peut être fait main libre ou en utilisant un manipulateur micro. Puis restaurer morceau cuticule sur la capsule céphalique et desserrer antennes. </li><li> Nourrir les abeilles avec une solution de saccharose à 30% avant de les stocker dans une affaire humidifié pendant au moins quatre heures ou une nuit à 20 ° C. </li></ol> L&#39;imagerie in vivo <ol><li> Empêcher les mouvements de l&#39;ex abeille avec un petit morceau d&#39;éponge qui est pressée contre le ventre et fixe avec un clip ou une bande à la chambre d&#39;enregistrement. </li><li> Pour empêcher le mouvement du cerveau résultant du pompage de l&#39;œsophage, couper une petite incision dans la cuticule au-dessus du labrum et tirez soigneusement l&#39;œsophage et de ses environs des structures solides pour le mettre sous tension, sans endommager l&#39;œsophage 4. Couvrir avec deux de silicone composant. </li><li> Faire des trous avec une aiguille et insérez électrodes en fil pour l&#39;enregistrement de M17. </li><li> Retirer morceau cuticule, la trachée et des glandes au-dessus du cerveau. Suce heamolymph intérieur de la capsule tête avec un morceau de papier. </li><li> Remplissez la capsule céphalique avec deux de silicone composant. Il est important que le cerveau est complètement couvert. </li><li> Abeilles lieu sur scène microscope et placer une goutte d&#39;eau sur la surface de la silicone, de plonger l&#39;objectif du microscope trempette dans la gouttelette. Focus sur les neurones colorés. </li></ol> Odeur et la stimulation d&#39;enregistrement des signaux Olfactomètre: Nous utilisons un ordinateur contrôlé, la stimulation périphérique personnalisé construit ou «olfactomètre» comme décrit précédemment par Galizia et Vetter 3. Les odeurs sont diluées dans un flux d&#39;air constant orienté vers les antennes. Toute relance se compose d&#39;une impulsion de l&#39;odeur de l&#39;air l&#39;odeur 3s 0.2ml saturé. Le protocole de stimulation peut être réglé dans le TILLVision logiciel d&#39;enregistrement. Microscope et imagerie mis en place: Nous utilisons un microscope à fluorescence Zeiss. Les images sont enregistrées à 25 ° C avec un taux d&#39;échantillonnage de 5 Hz en utilisant un dispositif d&#39;imagerie photonique TILL-monté sur le microscope. Signaux calciques sont enregistrées grâce à un X60, Olympus 0,9 W DIP objectif avec une caméra CCD Imago (640x480 pixels, 4X binned sur puce à 160x120 pixels). Fura-2 est excité par une lumière monochromatique de longueur d&#39;onde 340 et 380nm pour les mesures ratiométrique. La fluorescence est détectée à travers un miroir dichroïque 410 nm et un filtre passe 440 nm de long. Les paramètres pour l&#39;enregistrement sont définis dans le logiciel d&#39;enregistrement. Chaque mesure dure 10s et le temps d&#39;exposition pour les deux longueurs d&#39;onde peut être ajustée à l&#39;intensité de coloration dans chaque préparation. M17 enregistrement: Le muscle rapporteur du labium (M17) est enregistrée de façon extracellulaire de surveiller les réponses comportementales liées à l&#39;apprentissage, c&#39;est à dire le 5 p., 6. Injecter un fil de cuivre dans le muscle près de la bouche de pièces. Injecter électrode de masse dans l&#39;œil. Les potentiels musculaires sont amplifiés avec une version personnalisée de pré-amplificateur, numérisés et stockés sur un ordinateur. Le début du stimulus est déclenchée par l&#39;olfactomètre. Analyse des données <ol><li> Pendant l&#39;expérience: Les enregistrements du signal de calcium sont introduits dans un ordinateur et stockées. Le logiciel d&#39;enregistrement TILLVision permet une inspection initiale des signaux, comme le calcul du rapport entre les longueurs d&#39;onde (340nm/380nm) et le calcul des deltaF (soustraction de base). </li><li> Traitement de l&#39;image est réalisée avec la coutume des programmes écrits en langage IDL. Calculer le ratio de Ca 2 + signaux de 340nm et 380nm mesures pour chaque pixel. Déterminer fond de fluorescence (F) en moyenne plus de cadres avant le stimulus et soustraire au signal ratiométrique (deltaF). </li><li> Pour définir la région d&#39;intérêt (ROI): Calculer moyenne des cadres lors de la relance et appliquer un filtre passe-bas (3X3px). Convertir une échelle de gris à l&#39;échelle de couleurs fausses. Déterminer l&#39;activité dans les structures neuronales comme des taches d&#39;activité dans les images en fausses couleurs et de définir comme un retour sur investissement. </li><li> Calculer la dynamique temporelle des régions actives en faisant la moyenne des pixels dans la ROI sur toutes les trames sans aucun filtrage ou de correction. </li></ol> En collaboration avec le dextrane Fura-2, nous remplissons les neurones avec le colorant rhodamine fixable dextrane (&quot;mini-ruby&quot;). Les deux colorants ont le même poids moléculaire, ils doivent être co-localisés dans les neurones. Imagerie à grand champ ne permet que la résolution spatiale très limitée, il est donc recommandé d&#39;étudier les structures tachés après l&#39;expérience en utilisant la microscopie confocale à balayage. <ol><li> Après les mesures physiologiques, disséquer les cerveaux et les fixer. </li><li> Rincer avec du PBS et déshydrater par étapes dans l&#39;éthanol et le salicylate de méthyle dans les claires. </li><li> Pour la microscopie confocale intégrer cerveau en salicylate de méthyle. </li><li> Nous utilisons un Leica TCS SP2 microscope. Longueur d&#39;onde d&#39;excitation est 543nm. </li><li> Nous scannons le cerveau avec un air ou un objectif à immersion et de comparer les structures colorées avec les données d&#39;imagerie. </li></ol> Les résultats représentatifs: Si la coloration et la préparation a été réussie, le signal à l&#39;échelle de fausses couleurs, par exemple dans le corps du lobe des champignons neurones alpha extrinsèques, pourrait ressembler à la figure 1. La réponse comportementale (PER) peut être calculée comme la fréquence pic lors de la relance moins fréquence pic avant le stimulus (figure 2). Les structures tachés peuvent être étudiés plus en détail à l&#39;aide d&#39;un microscope confocal (figure 3). <img src="/files/ftp_upload/1353/figure1_new.png" alt="Figure 1" /> Figure 1: Représentant des signaux d&#39;imagerie calcique (stimulation avec heptanal). A: région imagée du cerveau d&#39;abeille. B: Raw image fluorescente comme vu à travers un objectif à immersion X20 longueur d&#39;onde d&#39;excitation 380nm, l&#39;alpha-lobe du corps champignon est décrite en violet. Dye a été injecté dans la jante ventro-latérale des lobes alpha-tacher neurones extrinsèques. C: l&#39;image en fausses couleurs à partir du signal moyenne, deltaF (340/380), lors de la relance trois secondes; alpha du lobe décrites en violet, sélectionnés ROI bordées de noir. D: Dynamique temporelle du signal calcique en réponse à la stimulation d&#39;odeurs, de signal moyen de la région d&#39;intérêt, comme indiqué dans C. E: Dynamique temporelle du signal calcique en réponse à la stimulation de saccharose de l&#39;IPSI et contra-latérale antenne respectivement à la RSI indiquée en C. <img src="/files/ftp_upload/1353/figure2_new.png" alt="" /> Figure 2: Représentant M17 réponse. Après conditionnement olfactif de l&#39;abeille s&#39;étend la trompe lorsque l&#39;odeur est présenté sans récompense. La ligne rouge indique relance odeur. En médaillon: PER de l&#39;abeille. <img src="/files/ftp_upload/1353/figure3_new.png" alt="" /> Figure 3: la pile d&#39;images représentant. Après l&#39;expérience d&#39;imagerie du cerveau a été disséqué. Le neurone de la figure 1 est scanné à l&#39;aide d&#39;un microscope confocal avec un objectif X20 immersion dans l&#39;huile, l&#39;alpha-lobe décrites en violet.

<ol>
	<li>Joerges, J., K&#252;ttner, A., Galizia, C. G., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representations+of+odors+and+odor+mixtures+visualized+in+the+honeybee+brain.">Representations of odors and odor mixtures visualized in the honeybee brain.</a> Nature. 387 (6630), 285-288 (1997).</li><li>Rehder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+honeybee's+proboscis+reflex+by+electromyiographic+recordings.">Quantification of the honeybee's proboscis reflex by electromyiographic recordings.</a> J. Insect Physiol. 33, 501-507 (1987).</li><li>Galizia, C. G., Vetter, R. S., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Methods+for+Analyzing+Odor-Evoked+Activity+in+the+Insect+Brain.">Optical Methods for Analyzing Odor-Evoked Activity in the Insect Brain.</a> Methods in Insect Sensory Neuroscience. , 345-388 (2004).</li><li>Mauelshagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+correlates+of+olfactory+learning+paradigms+in+an+identified+neuron+in+the+honeybee+brain.">Neural correlates of olfactory learning paradigms in an identified neuron in the honeybee brain.</a> J Neurophysiol. 69 (2), 609-625 (1993).</li><li>Kuwabara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bildung+des+bedingten+Reflexes+von+Pavlovs+Typus+bei+der+Honigbiene+Apis+mellifica,+Hokaido+Univ.">Bildung des bedingten Reflexes von Pavlovs Typus bei der Honigbiene Apis mellifica, Hokaido Univ.</a> Zool. J. Sci. 13, 458-464 (1957).</li><li>Bitterman, M. E., Menzel, R., Fietz, A., Schafer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+conditioning+of+proboscis+extension+in+honeybees+(Apis+mellifera.">Classical conditioning of proboscis extension in honeybees (Apis mellifera.</a> Journal of Comparative Psychology. 97 (2), 107-119 (1983).</li></ol>

Dans cette présentation, nous avons traversé toutes les mesures nécessaires pour enquêter dans les signaux calciques in vivo chez l&#39;abeille. Nous avons appliqué cette technique à des neurones du corps de champignons, mais l&#39;imagerie peut être effectuée sur tous les neurones pour lesquels une technique de coloration peut être établie. Nous nous sommes concentrés sur les neurones de la voie olfactive de l&#39;abeille. Les expériences sont toutes effectuées dans une chambre noire. La ...

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<td>low melting point hard wax Deiberit 502</td>
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<td>Dr. B&ouml;hme & Sch&ouml;ps Dental GmbH</td>
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<td>FURA-2 dextran potassium salt, 10 000 MW</td>
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<td>tetramethylrhodamine dextran 10 000 MW</td>
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in vivo ca2+- imaging of mushroom body neurons during olfactory learning in the honey bee

L&#39;in vivo et semi-en préparation pour l&#39;imagerie in vivo de calcium a été développé dans notre laboratoire par Joerges, Kuttner et Galizia il ya dix ans, pour mesurer l&#39;activité d&#39;odeur évoquée dans le lobe antennaire 1. Dès lors, il a été constamment affiné et appliqué à neuropiles différents dans le cerveau de l&#39;abeille. Ici, nous décrivons la préparation actuellement utilisés dans le laboratoire pour mesurer l&#39;activité des neurones du corps en utilisant un champignon couplé dextrane sensible au calcium colorant (Fura-2). Nous rétrograde tache neurones du corps de champignons en injectant un colorant dans leurs axones ou région soma. Nous nous concentrons sur la réduction de l&#39;envahissement, de réaliser une préparation dans laquelle il est encore possible de former l&#39;abeille en utilisant PER conditionné. Nous sommes en mesure de surveiller et de quantifier la réponse comportementale par l&#39;enregistrement électro-myograms du muscle qui contrôle le PER (M17) 2. Après l&#39;expérience des structures imagées physiologiques sont étudiés plus en détail en utilisant la microscopie confocale à balayage à l&#39;adresse de l&#39;identité des neurones.

Watch this Scientific Journal Video about In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee at JoVE.com

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Les abeilles peuvent être conditionnés dans un paradigme d&#39;apprentissage olfactif appétitif (PER-conditionné). L&#39;utilisation des odeurs comme stimuli, nous avons établi une méthode dans laquelle le comportement est enregistré pendant l&#39;imagerie calcique simultanée est utilisée pour mesurer l&#39;activité d&#39;odeur évoquée dans les neurones du corps de champignons<em&gt; In vivo</em&gt;.

In vivo Ca 2 + - Imagerie des neurones du corps champignons Pendant l&#39;apprentissage olfactif dans le Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

in-vivo-ca2-imaging-mushroom-body-neurons-during-olfactory-learning

Research

JoVE Journal

Biology

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

13.9K vues.  Freie Universität Berlin. Les abeilles peuvent être conditionnés dans un paradigme d'apprentissage olfactif appétitif (PER-conditionné). L'utilisation des odeurs comme stimuli, nous avons établi une méthode dans laquelle le comportement est enregistré pendant l'imagerie calcique simultanée est utilisée pour mesurer l'activité d'odeur évoquée dans les neurones du corps de champignons In vivo.

Vidéo: In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

Olfactive tests comportementaux chez la souris adulte

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

Recherche

Biologie

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

Micro-drive tableau pour les maladies chroniques In vivo Enregistrement: Disque de fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential amplitude of action potential signatures over the channels of a tetrode allows for the isolation of single-unit activity from multi-unit signals.  The ability to precisely control the stereotaxic location and depth of the tetrode is critical for studying coordinated neural activity across brain regions.  In combination with a micro-drive array, it is possible to achieve precise placement and stable control of many tetrodes over the course of days to weeks.  In this protocol, we demonstrate how to fabricate and condition tetrodes using basic tools and materials, install the tetrodes into a multi-drive tetrode array for chronic in-vivo recording in the rat, make ground wire connections to the micro-drive array, and attach a protective cone onto the micro-drive array in order to protect the tetrodes from physical contact with the environment.



Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.

Micro-drive tableau pour les maladies chroniques In vivo Enregistrement: Assemblée Tétrode

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Techniques d&#39;imagerie Ca 2 + Signalisation dans le sperme humain

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Analyse rapide de la génétique des épithélio-mésenchymateuse signalisation lors de la régénération des cheveux

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivre imagerie de dédouanement cellulaire par apoptose au cours<em&gt; Drosophila</em&gt; Embryogenèse

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.

In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence.

Obtenir des échantillons avec ralenti accéléré et inversé vieillissement dans le Honey Bee Modèle

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

Méthodes de comparaison des éléments nutritifs dans gelée royale faites par africanisées et Honey Bees européenne et les effets sur hémolymphe Les titres de protéines

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagerie Ca locale<sup&gt; 2+</sup&gt; Signaux de cellules de mammifères cultivées

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Haute résolution Time-lapse d&#39;imagerie et d&#39;analyse automatisée des microtubules Dynamics Living ombilicale humaine Cellules endothéliales de veine

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...