Questo lavoro è finanziato dalla DFG.

Bee Preparazione e Back-riempiono <ol><li> Cattura api bottinatrici in arnia e freddo in ghiaccio per immobilizzazione. </li><li> Montaggio in plexiglass registrazione camere 3. Fissare gli occhi e torace alle pareti della camera di registrazione con la cera a basso punto di fusione dura. </li><li> Tirare in vetro capillari, comunemente usate per elettrofisiologia e rompere alla punta ad ottenere un diametro punta di ca. 10μm. Coprire punta del capillare con pasta colorante. Pasta colorante è costituito da una miscela 10:01 di Fura-2 destrano e lisina risolvibile tetrametilrodamina destrano. </li><li> Per la procedura di colorazione immobilizzare antenne con minuzie o n-eicosane. Rimuovere un pezzo di cuticola al di sopra del cervello e spingere ghiandole e trachea al lato per consentire l&#39;accesso al corpo dei funghi. </li><li> Iniettare capillari in nessuna regione del soma o assonale dei neuroni del corpo funghi. Questo può essere fatto a mano libera o utilizzando un micro-manipolatore. Quindi ripristinare pezzo cuticola sulla capsula testa e allentare antenne. </li><li> Nutrire le api con una soluzione di saccarosio al 30% prima di riporlo in un caso umidificato per almeno quattro ore o per tutta la notte a 20 ° C. </li></ol> Imaging in vivo <ol><li> Prevenire i movimenti dei es. api con un piccolo pezzo di spugna che viene premuto contro l&#39;addome e fissa con una clip o nastro alla camera di registrazione. </li><li> Per impedire il movimento del cervello risultante dal pompaggio dell &#39;esofago, tagliate una piccola incisione nella cuticola sopra il labbro ed estrarre con attenzione l&#39;esofago e le sue solide strutture circostanti per metterla in tensione, senza danneggiare l&#39;esofago 4. Coprire con due componenti in silicone. </li><li> Eseguire fori con un ago ed inserire elettrodi a filo per la registrazione di M17. </li><li> Rimuovere pezzo cuticola, trachea e le ghiandole sopra il cervello. Succhiare heamolymph all&#39;interno della capsula testa con un pezzo di carta. </li><li> Riempire la capsula testa con due componenti in silicone. E &#39;importante che il cervello è completamente coperto. </li><li> Ape posto sul palco microscopio e una goccia d&#39;acqua sulla superficie del silicone per immergere l&#39;obiettivo del microscopio tuffo nella goccia. Focus su neuroni macchiato. </li></ol> Odore stimolazione e registrazione di segnali Olfattometro: Usiamo un controllo del computer, dispositivo di stimolazione personalizzati costruiti o &quot;olfattometro&quot; come descritto in precedenza da Galizia e Vetter 3. Gli odori sono diluiti in un flusso d&#39;aria costante diretto verso le antenne. Ogni stimolo consiste in un impulso 3s odore di aria satura dell&#39;odore 0,2 ml. Il protocollo di stimolazione può essere impostato nel TILLVision software di registrazione. Microscopio Imaging e set-up: Noi usiamo un microscopio a fluorescenza Zeiss. Le immagini vengono registrate a 25 ° C con un tasso di campionamento di 5 Hz utilizzando un AL-Photonics dispositivo di imaging montato sul microscopio. Segnali di calcio sono registrati attraverso un X60, 0,9 W Olympus tuffo obiettivo con una fotocamera CCD Imago (640X480 pixel, 4X cestinate su chip a 160x120 pixel). Fura-2 è eccitato con luce monocromatica di 340 e 380 nm di lunghezza d&#39;onda per misure raziometrico. Fluorescenza viene rilevata attraverso uno specchio dicroico 410nm e 440nm un filtro passaggio lungo. I parametri per la registrazione sono impostate nel software di registrazione. Ogni misura ha una durata di 10 secondi e tempo di esposizione per le due lunghezze d&#39;onda può essere regolata per l&#39;intensità di colorazione in ogni preparazione. M17 registrazione: Il muscolo goniometro del labium (M17) è registrato extracellulare di monitorare le risposte comportamentali legati all&#39;apprendimento, cioè il PER 5, 6. Iniettare un filo di rame nel muscolo vicino al parti della bocca. Iniettare elettrodo di terra negli occhi. I potenziali muscolari sono amplificate con una generazione personalizzata pre-amplificatore, digitalizzati e archiviati su un computer. Insorgenza stimolo è innescato dal olfattometro. Analisi dei dati <ol><li> Durante l&#39;esperimento: Le registrazioni del segnale calcio sono immessi in un computer e memorizzati. Il TILLVision software di registrazione permette una ispezione iniziale dei segnali, come il calcolo del rapporto tra le lunghezze d&#39;onda (340nm/380nm) e calcolo di deltaF (sottrazione di base). </li><li> L&#39;elaborazione delle immagini viene eseguita con programmi personalizzati scritti in IDL. Calcolare il rapporto di Ca 2 + segnali da 340nm e 380nm misure per ogni pixel. Determinare gli fluorescenza (F) da una media di oltre fotogrammi prima lo stimolo e sottrarre dal segnale raziometrico (deltaF). </li><li> Per definire Regione di interesse (ROI): Calcolare media dei fotogrammi durante lo stimolo e applicare un filtro passa-basso (3X3px). Convertire in scala di grigi a scala di falsi colori. Determinare l&#39;attività in strutture neuronali come macchie attività nelle immagini in falsi colori e definire come il ROI. </li><li> Calcolare la dinamica temporale delle regioni attive facendo la media dei pixel nella ROI su tutti i fotogrammi, senza alcun filtro o correzione. </li></ol> Insieme al Fura-2 destrano riempiamo i neuroni con la risolvibile colorante rodamina destrano (&quot;mini rubino&quot;). Entrambi i coloranti hanno lo stesso peso molecolare, che devono essere co-locati nei neuroni. Grande campo di imaging permette solo la risoluzione spaziale molto limitata, quindi si raccomanda di indagare le strutture macchiate dopo l&#39;esperimento usando la microscopia confocale a scansione. <ol><li> Dopo le misurazioni fisiologiche, sezionare il cervello e fissare. </li><li> Sciacquare in PBS e disidratare in etanolo graduale e chiaro in salycilate metile. </li><li> Per la microscopia confocale incorporare cervello in salycilate metile. </li><li> Usiamo un microscopio Leica TCS SP2. Lunghezza d&#39;onda di eccitazione è 543nm. </li><li> Abbiamo la scansione del cervello con l&#39;aria o l&#39;immersione obiettivo di petrolio e confrontare le strutture colorate con i dati di imaging. </li></ol> Rappresentante dei risultati: Se la colorazione e la preparazione ha avuto successo, il segnale in scala di falsi colori, ad esempio in funghi corpo alfa neuroni del lobo estrinseca, potrebbe apparire come in figura 1. La risposta comportamentale (PER) può essere calcolato come frequenza picco durante lo stimolo meno frequenza di picco prima lo stimolo (Figura 2). Le strutture colorate possono essere studiati in dettaglio utilizzando un microscopio confocale (Figura 3). <img src="/files/ftp_upload/1353/figure1_new.png" alt="Figura 1" /> Figura 1: Rappresentante segnali Imaging di calcio (stimolazione con heptanal). R: Imaged regione del cervello delle api. B: prime immagini fluorescenti, come visto attraverso un tuffo X20 obiettivo alla lunghezza d&#39;onda di eccitazione 380 nm, l&#39;alfa-lobo del corpo dei funghi è delineato in viola. Colorante è stato iniettato nel ventro-laterale bordo della alfa-lobi a macchia neuroni estrinseci. C: immagine a colori falsi derivato dalla media del segnale, deltaF (340/380), durante lo stimolo tre secondi; alfa-lobo delineato in viola, selezionati ROI delineati in nero. D: dinamica temporale del segnale del calcio in risposta alla stimolazione odore, segnale media della regione di interesse, come indicato in C. E: la dinamica temporale del segnale del calcio in risposta alla stimolazione del saccarosio ipsi-e contra-laterali antenna rispettivamente al ROI indicato in C. <img src="/files/ftp_upload/1353/figure2_new.png" alt="" /> Figura 2: Rappresentante M17 risposta. Dopo il condizionamento olfattivo l&#39;ape estende la proboscide, quando l&#39;odore è presentato senza ricompensa. Linea rossa indica stimolo odore. Inset: PER delle api. <img src="/files/ftp_upload/1353/figure3_new.png" alt="" /> Figura 3: serie di immagini Rappresentante. Dopo l&#39;esperimento di imaging del cervello è stato sezionato. Il neurone di figura 1 viene sottoposto a scansione utilizzando un microscopio confocale con un obiettivo ad immersione olio X20, l&#39;alfa-lobo delineato in viola.

<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 questa presentazione abbiamo vissuto tutti i passaggi per indagare nei segnali di calcio in vivo l&#39;ape. Abbiamo applicato questa tecnica ai neuroni del corpo dei funghi, ma l&#39;imaging può essere fatto su tutti i neuroni per cui una tecnica di colorazione può essere stabilita. Ci siamo concentrati sui neuroni nel percorso olfattivo delle api. Gli esperimenti sono tutti eseguiti in una camera oscura. Il set up è illuminato con una luce onda lunga che si trova fuori dal campo della percezione...

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in vivo ca2+- imaging of mushroom body neurons during olfactory learning in the honey bee

In vivo e semi-in preparazione per l&#39;imaging in vivo di calcio è stato sviluppato nel nostro laboratorio da Joerges, Kuttner e Galizia oltre dieci anni fa, per misurare l&#39;attività odore evocato nel lobo antennale 1. Da allora in poi, è stato continuamente perfezionato e applicato a diversi neuropiles nel cervello delle api. Qui, descriviamo la preparazione attualmente in uso in laboratorio per misurare l&#39;attività nei neuroni del corpo dei funghi con un accoppiato destrano calcio-sensibili colorante (Fura-2). Noi retrogrado macchia neuroni corpo funghi, iniettando colorante nella loro assoni o regione soma. Ci si concentra sulla riduzione della invasività, per ottenere una preparazione in cui è ancora possibile formare l&#39;ape utilizzando PER condizionata. Siamo in grado di monitorare e quantificare la risposta comportamentale di registrazione elettro-myograms dal muscolo che controlla il PER (M17) 2. Dopo l&#39;esperimento fisiologico delle strutture ripreso sono studiati in dettaglio utilizzando la microscopia confocale a scansione per affrontare l&#39;identità dei neuroni.

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

Le api possono essere condizionate in un paradigma di apprendimento olfattivo appetitivo (PER condizionata). Utilizzando gli odori come stimoli, abbiamo stabilito un metodo in cui il comportamento viene registrato contemporaneamente Imaging calcio è utilizzato per misurare l&#39;attività odore evocato nei neuroni del corpo dei funghi<em&gt; In vivo</em&gt;.

In vivo Ca 2 + - Imaging di neuroni Corpo funghi durante l&#39;apprendimento olfattivo nel 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 Views.  Freie Universität Berlin. Le api possono essere condizionate in un paradigma di apprendimento olfattivo appetitivo (PER condizionata). Utilizzando gli odori come stimoli, abbiamo stabilito un metodo in cui il comportamento viene registrato contemporaneamente Imaging calcio è utilizzato per misurare l'attività odore evocato nei neuroni del corpo dei funghi In vivo.

Video: 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.

Olfattivo test comportamentali nel topo adulto

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

Ricerca

Biologia

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 array per la cronica In vivo</emRegistrazione>: Drive 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 array per la cronica In vivo</emRegistrazione>: tetrodo Assemblea

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.

Tecniche di Imaging Ca 2 + Segnalazione di sperma umano

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.

Rapida analisi genetica di epitelio-mesenchimale segnalazione durante la rigenerazione dei capelli

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.

Vivere Imaging di apoptotica delle cellule libera nel corso<em&gt; Drosophila</em&gt; Embriogenesi

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.

Ottenere campioni con rallentato, accelerato e invertito l&#39;invecchiamento in Honey Bee Modello

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.

Metodi per confrontare nutrienti in beebread Made by africanizzate e miele europeo api e gli effetti sulla I titoli emolinfa Protein

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 locale Ca<sup&gt; 2+</sup&gt; Segnali in cellule di mammifero coltivate

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.

Ad alta risoluzione Time-lapse imaging e analisi automatizzata dei microtubuli Dynamics in Living ombelicale umano cellule endoteliali Vena

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