Diese Arbeit wird von der DFG gefördert.

Bee Vorbereitung und Zurück-fill <ol><li> Fangen Honigbiene Sammlerinnen bei hive-und Chill auf Eis Immobilisierung. </li><li> Berg in Plexiglas Aufnahme Kammern 3. Fix Augen und Thorax an den Wänden der Aufnahme Kammer mit niedrigem Schmelzpunkt Hartwachs. </li><li> Ziehen von Glaskapillaren als gewöhnlich für Elektrophysiologie und brechen an der Spitze zu einer Spitze Durchmesser von ca. erhalten. 10 um. Abdeckung Spitze der Kapillare mit Farbpaste. Dye Paste besteht aus einem 10:1-Gemisch von Fura-2 Dextran und Lysin feststellbar Tetramethylrhodamin Dextran. </li><li> Für die Färbung immobilisieren Antennen mit Kleinigkeiten oder n-Eicosan. Nehmen Sie ein Stück Epidermis über dem Gehirn und schieben Drüsen und die Luftröhre zur Seite, um Zugang zu den Pilzkörper ermöglichen. </li><li> Inject Kapillaren entweder in die Soma oder axonalen Region Pilzkörper Neuronen. Dies kann frei Hand oder mit einem Mikro-Manipulator. Dann wieder Nagelhaut Stück auf Kopfkapsel und lockern Antennen. </li><li> Feed Bienen mit 30% Saccharose-Lösung vor dem Speichern in einem befeuchteten Fall für mindestens vier Stunden oder über Nacht bei 20 ° C. </li></ol> In-vivo-Imaging <ol><li> Verhindern Bewegungen der Biene z. B. mit einem kleinen Stück Schwamm, der gegen den Bauch gedrückt und fixiert mit einem Clip oder Band, um die Aufnahme Kammer. </li><li> Um zu verhindern, Bewegung des Gehirns, die sich aus Pumpen der Speiseröhre, schneiden einen kleinen Einschnitt in die Kutikula über dem Labrum und ziehen Sie vorsichtig die Speiseröhre und den umliegenden festen Strukturen, um es unter Spannung gesetzt, ohne die Speiseröhre 4. Deckel mit Zwei-Komponenten-Silikon. </li><li> Machen Sie Löcher mit einer Nadel und stecken Drahtelektroden für die Aufnahme von M17. </li><li> Entfernen Nagelhaut Stück, Luftröhre und Drüsen oberhalb des Gehirns. Saugen heamolymph im Kopf Kapsel mit einem Stück Papier. </li><li> Füllen Sie den Kopf Kapsel mit Zwei-Komponenten-Silikon. Es ist wichtig, dass das Gehirn vollständig bedeckt ist. </li><li> Legen Sie Biene auf Mikroskop-Bühne und geben Sie einen Tropfen von Wasser auf der Oberfläche des Silikons zu den Dip Objektiv des Mikroskops in den Tropfen eintauchen. Konzentrieren Sie sich auf gefärbte Neuronen. </li></ol> Odor Stimulation und Aufzeichnung von Signalen Olfaktometer: Wir verwenden einen Computer gesteuert, custom built Stimulationsgerät oder &quot;Olfaktometer&quot;, wie zuvor von Galizia und Vetter 3 beschrieben. Gerüche werden in einen konstanten Luftstrom in Richtung der Antennen gerichtet verdünnt. Jeder Reiz besteht aus einem 3-Fettsäuren Geruch Puls von 0.2ml Geruch gesättigte Luft. Die Stimulation Protokoll kann in der Aufnahme-Software TILLVision eingestellt werden. Mikroskop-und Imaging-Set-up: Wir verwenden ein Zeiss Fluoreszenzmikroskop. Die Bilder werden bei 25 ° C mit einer Sampling-Rate von 5 Hz mit einer TILL-Photonics Imaging-Gerät auf dem Mikroskop montiert aufgezeichnet. Calcium-Signale werden durch eine X60, 0,9 W Olympus dip Ziel mit einem Imago CCD-Kamera (640x480 Pixel, 4fach-on-Chip auf 160x120 Pixel Binning) aufgezeichnet. Fura-2 ist mit monochromatischem Licht von 340 und 380nm Wellenlänge für ratiometrische Messungen aufgeregt. Die Fluoreszenz wird durch einen 410 nm dichroitischen Spiegel und einem 440nm Langpassfilter erkannt. Die Parameter für die Aufnahme in die Aufnahme-Software eingestellt. Jede Messung dauert 10s und Belichtungszeit für die beiden Wellenlängen, die Intensität der Färbung in jedem Präparat eingestellt werden kann. M17-Aufnahme: Die Winkelmesser Muskel des Labium (M17) extrazellulär ist es, die Verhaltensreaktionen in Bezug auf das Lernen Monitor aufgezeichnet, dh die PRO 5, 6. Inject einen Kupferdraht in den Muskel in der Nähe der Mundwerkzeuge. Inject Masseelektrode ins Auge. Der Muskel Potentiale werden mit einem benutzerdefinierten Build-Vorverstärker verstärkt, digitalisiert und gespeichert auf einem Computer. Beginn des Stimulus wird durch das Olfaktometer ausgelöst. Data Analysis <ol><li> Während des Experiments: Die Aufnahmen der Calcium-Signal in einen Computer eingespeist und gespeichert werden. Die Aufnahme-Software ermöglicht TILLVision einer Erstprüfung der Signale, wie die Berechnung des Verhältnisses zwischen den Wellenlängen (340nm/380nm) und Berechnung von deltaf (Subtraktion des Ausgangswertes). </li><li> Bildverarbeitung mit eigens geschriebenen Programmen in IDL durchgeführt. Berechnen Verhältnis von Ca 2 +-Signale von 340nm und 380nm Messungen für jedes Pixel. Bestimmen Sie Hintergrundfluoreszenz (F) durch Mittelung über Frames vor dem Reiz und subtrahieren von der ratiometrische Signal (deltaf). </li><li> Um Region of Interest (ROI) zu definieren: Berechnen Sie Mittelwert von Frames während der Reiz und wenden Sie einen Tiefpassfilter (3X3px). Convert Graustufen auf false Farbskala. Bestimmen Aktivität in neuronalen Strukturen als Aktivität Spots in falsche Farbbilder und definieren als ROI. </li><li> Berechnen Sie die zeitliche Dynamik der aktiven Bereiche durch Mittelung der Pixel in der ROI über alle Frames ohne Filterung oder Korrektur. </li></ol> Zusammen mit der Fura-2 Dextran füllen wir die Neuronen mit den fixierbaren Farbstoff Rhodamin Dextran (&quot;Mini-ruby&quot;). Beide Farbstoffe haben das gleiche Molekulargewicht, sie sollten in den Neuronen co-lokalisiert werden. Weitwinkel-Bildgebung ermöglicht nur eine sehr begrenzte räumliche Auflösung, daher ist es empfehlenswert, die gefärbten Strukturen nach dem Experiment mit Hilfe der konfokalen Mikroskopie zu untersuchen. <ol><li> Nach physiologische Messungen, sezieren Gehirn und zu fixieren. </li><li> Spülen in PBS und schrittweise entwässern in Ethanol und klar in Methyl Salycilat. </li><li> Für die konfokale Mikroskopie einbetten Gehirn in Methyl Salycilat. </li><li> Wir verwenden ein Leica TCS SP2-Mikroskop. Anregungswellenlänge 543nm. </li><li> Wir scannen das Gehirn mit einem Luft-oder Ölimmersionsobjektiv und vergleichen Sie die gefärbten Strukturen mit den Bilddaten. </li></ol> Repräsentative Ergebnisse: Wenn die Färbung und die Vorbereitung erfolgreich war, wird das Signal in falsche Farbskala, zum Beispiel in Pilzkörper alpha lobe extrinsischen Neuronen, könnte wie in Abbildung 1 aussehen. Die Verhaltensreaktion (PER) als Spike-Frequenz während der Reiz minus Spikefrequenz vor dem Reiz (Abbildung 2) berechnet werden. Die gefärbten Strukturen können in größerem Detail mit einem konfokalen Mikroskop (Abbildung 3) untersucht werden. <img src="/files/ftp_upload/1353/figure1_new.png" alt="Abbildung 1" /> Abbildung 1: Repräsentative Calcium Imaging-Signale (Stimulation mit Heptanal). A: abgebildeten Bereich der Biene Gehirn. B: Raw Fluoreszenzbild wie durch ein X20 dip Ziel bei 380nm Anregungswellenlänge gesehen, ist die alpha-Lappen der Pilzkörper in lila dargestellt. Dye wurde in den ventro-lateralen Rand des alpha-Lappen injiziert, um extrinsische Neurone Fleck. C: Falschfarben-Bild aus dem durchschnittlichen Signal, deltaf (340/380), während die drei zweiten Reiz gewonnen; alpha-Lappen in lila dargestellt, erläutert ausgewählten ROI in schwarz. D: Zeitliche Dynamik der Kalzium-Signal in Reaktion auf Duftpuls, durchschnittliche Signal aus der Region von Interesse, da in C. E angegeben: Zeitliche Dynamik der Kalzium-Signal in Reaktion auf Saccharose Stimulation der ipsi-und kontralateralen Antenne bzw. der ROI angegeben in C. <img src="/files/ftp_upload/1353/figure2_new.png" alt="" /> Abbildung 2: Repräsentative M17 Reaktion. Nach olfaktorischen Konditionierung der Biene erweitert den Rüssel, wenn der Geruch ohne Belohnung wird vorgestellt. Rote Linie zeigt Geruch Reiz. Inset: PER der Honigbiene. <img src="/files/ftp_upload/1353/figure3_new.png" alt="" /> Abbildung 3: Repräsentative Bildstapel. Nach dem Imaging-Experiment wurde das Gehirn seziert. Das Neuron aus Abbildung 1 wird gescannt mit einem konfokalen Mikroskop mit einem X20 Ölimmersionsobjektiv, alpha-Lappen in lila dargestellt.

<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>

In dieser Präsentation haben wir alle Schritte gegangen, um in vivo Calcium-Signale in der Honigbiene zu untersuchen. Wir wandten diese Technik auf Pilzkörper Neuronen, sondern Bildgebung kann auf alle Neurone, für die eine Maltechnik hergestellt werden kann getan werden. Wir haben auf Neuronen im olfaktorischen Weg der Honigbiene konzentriert. Die Experimente sind in einer Dunkelkammer durchgeführt. Die Einrichtung ist mit einem langwelligen Licht, das aus Lügen der optischen Wahrnehmung Bereich der Biene...

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<td>low melting point hard wax Deiberit 502</td>
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in vivo ca2+- imaging of mushroom body neurons during olfactory learning in the honey bee

Die in-vivo-und semi-in vivo Vorbereitung für Calcium Imaging hat in unserem Labor durch Joerges, Küttner und Galizia entwickelte vor über zehn Jahren, um Gerüche zu evozierte Aktivität im Antennallobus 1 zu messen. Von da an wurde kontinuierlich weiterentwickelt und an verschiedenen neuropiles in der Biene Gehirn. Hier beschreiben wir die Vorbereitungen derzeit im Labor verwendet werden, um Aktivität in Pilzkörper Neuronen mit einem Dextran gekoppelt Calcium-sensitiven Farbstoff (Fura-2) zu messen. Wir retrograd Fleck Pilzkörper Neuronen durch die Injektion von Farbstoff in ihre Axone oder soma Region. Wir konzentrieren uns auf die Verringerung der Invasivität zu konzentrieren, eine Vorbereitung, in denen es immer noch möglich, die Biene mit PER Klimaanlage Zug zu erreichen. Wir sind in der Lage zu überwachen und zu quantifizieren, die Verhaltensreaktion durch die Aufnahme elektro-myograms aus dem Muskel, die die PRO (M17) 2 steuert. Nach dem physiologischen Experiment die abgebildeten Strukturen sind in größerem Detail mit Hilfe der konfokalen Mikroskopie, die Identität der Neuronen-Adresse untersucht.

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

Bienen können in einer appetitive olfaktorischen Lernparadigma (PER-Konditionierung) konditioniert werden. Mit Gerüche als Stimuli, haben wir ein Verfahren, bei welchem ​​Verhalten aufgezeichnet, während gleichzeitig Calcium Imaging wird verwendet, um Geruchs-evozierte Aktivität im Pilzkörper Neuronen zu messen ist<em&gt; In vivo</em&gt;.

In vivo Ca 2 + - Imaging von Pilzkörper Neuronen während olfaktorischen Lernen in der Honigbiene

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 Aufrufe.  Freie Universität Berlin. Bienen können in einer appetitive olfaktorischen Lernparadigma (PER-Konditionierung) konditioniert werden. Mit Gerüche als Stimuli, haben wir ein Verfahren, bei welchem ​​Verhalten aufgezeichnet, während gleichzeitig Calcium Imaging wird verwendet, um Geruchs-evozierte Aktivität im Pilzkörper Neuronen zu messen ist In vivo.

Serie: 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.

Olfaktorische Behavioral Testing in der adulten Maus

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

Forschung

Biologie

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-Array für chronische 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 ...

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 for Imaging Ca 2 + Signaling in menschlichen Spermien

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.

Schnelle Genetische Analyse von epithelial-mesenchymale Signaling Während Haarregeneration

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.

Live-Imaging von apoptotischen Ausverkauf während<em&gt; Drosophila</em&gt; Embryogenese

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.

Beziehen Proben mit verlangsamt, Beschleunigte und Umge Aging in der Honig-Bienen-Modell

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.

Methoden zum Vergleich der Nährstoffe in Bienenbrot Hergestellt von Africanized und europäischen Honigbienen und die Auswirkungen auf die Hämolymphe Protein Titer

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.

Imaging Lokale Ca<sup&gt; 2+</sup&gt; Signale in kultivierten Säugetierzellen

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.

Hochauflösende Zeitraffer-Imaging und automatische Analyse von Dynamik der Mikrotubuli in lebenden menschlichen Endothelzellen aus der Nabelvene

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

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

Dissection und Beobachtung von Honey Bee Dorsal Schiff für Untersuchungen der Herzfunktion

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...