Este trabalho é financiado pela DFG.

Bee Preparação e Back-fill <ol><li> Forrageiras pegar mel de abelha em colmeia e frio no gelo para imobilização. </li><li> Montagem em Plexiglas gravação câmaras 3. Fixar os olhos e tórax para as paredes da câmara gravando com cera baixo ponto de fusão rígido. </li><li> Puxe capilares de vidro tão comumente utilizado para eletrofisiologia e quebrar na ponta para obter um diâmetro da ponta de ca. 10μm. Cobrir a ponta do capilar com pasta de corante. Colar Dye consiste de uma mistura 10:01 de Fura-2 dextran e fixable lisina tetrametilrodamina dextran. </li><li> Para o procedimento de coloração imobilizar antenas com minúcias ou n-eicosane. Retire um pedaço de cutícula acima do cérebro e glândulas empurrar e traquéia para o lado para permitir o acesso ao corpo de cogumelo. </li><li> Injetar capilares em qualquer soma ou região axonal dos neurônios do corpo de cogumelo. Isto pode ser feito a mão livre ou através de um manipulador micro. Em seguida, restaurar parte da cutícula na cápsula de cabeça e soltar antenas. </li><li> Alimentar as abelhas com solução de sacarose 30% antes de armazená-los em um caso umidificado por pelo menos quatro horas ou durante a noite a 20 ° C. </li></ol> In vivo de imagens <ol><li> Impedir os movimentos do exemplo da abelha com um pequeno pedaço de esponja que é pressionado contra o abdômen e fixada com um clipe ou fita para a câmara de gravação. </li><li> Para impedir o movimento do cérebro resultante de bombeamento do esôfago, corte uma pequena incisão na cutícula acima do labrum e retire cuidadosamente o esôfago e seu entorno estruturas sólidas para colocá-la sob tensão, sem danificar o esôfago 4. Cubra com duas silicone componente. </li><li> Faça furos com uma agulha e inserir eletrodos de arame para a gravação de M17. </li><li> Retire parte da cutícula, traquéia e glândulas acima do cérebro. Sugar heamolymph dentro da cápsula cabeça com um pedaço de papel. </li><li> Encher a cápsula cabeça com dois silicone componente. É importante que o cérebro está completamente coberto. </li><li> Abelha lugar no palco do microscópio e coloque uma gota de água sobre a superfície do silicone para imergir o objetivo mergulho do microscópio para a gota. Concentre-se em neurônios manchado. </li></ol> Estimulação odor e gravação de sinais Olfatômetro: Nós usamos um computador controlado, dispositivo de estimulação personalizado construído ou &quot;olfatômetro&quot;, como descrito anteriormente por Galizia e Vetter 3. Odores são diluídos em um fluxo constante de ar dirigido para as antenas. Qualquer estímulo consiste de um pulso de 3s odor de ar odor 0.2ml saturada. O protocolo de estimulação pode ser definido na TILLVision software de gravação. Microscópio e Imagem set-up: Nós usamos um microscópio de fluorescência Zeiss. As imagens são gravadas a 25 ° C com uma taxa de amostragem de 5 Hz usando uma TILL-Photonics dispositivo de imagem montado no microscópio. Sinais de cálcio são registrados através de um X60, 0,9 W Olympus mergulho objetivo com um CCD Imago câmera (640X480 pixels, 4X binned em chip para 160X120 pixels). Fura-2 está animado com a luz monocromática de 340 e 380nm de comprimento de onda para medições raciométrica. Fluorescência é detectada através de um espelho dicróico 410nm e um filtro passa-440nm de comprimento. Os parâmetros para a gravação são definidas no software de gravação. Cada medição dura 10s e tempo de exposição para os dois comprimentos de onda pode ser ajustado para a intensidade de coloração em cada preparação. M17 gravação: O músculo transferidor do lábio (M17) é gravado extracelularmente para monitorar as respostas comportamentais relacionados com a aprendizagem, ou seja, os 5 PER, 6. Injetar um fio de cobre no músculo perto da partes da boca. Injetar eletrodo terra no olho. Os potenciais musculares são amplificados com um costume construir pré-amplificador, digitalizadas e armazenadas em um computador. Início do estímulo é desencadeado pela olfatômetro. Análise de Dados <ol><li> Durante o experimento: As gravações do sinal de cálcio são alimentados em um computador e armazenado. O TILLVision software de gravação permite uma inspecção inicial dos sinais, como o cálculo da relação entre os comprimentos de onda (340nm/380nm) e cálculo de deltaF (subtração da linha de base). </li><li> Processamento de imagem é realizada com o costume programas escritos em IDL. Calcular proporção de Ca 2 + sinais de 340nm e 380nm medições para cada pixel. Determine fundo de fluorescência (F) pela média de mais de frames antes do estímulo e subtrair o sinal raciométrica (deltaF). </li><li> Para definir região de interesse (ROI): Calcular média de frames durante o estímulo e aplicar um filtro passa-baixa (3X3px). Converter em escala de cinza para escala de cores falsas. Determinar a atividade neuronal em estruturas como pontos de atividade em imagens de cores falsas e definir como ROI. </li><li> Calcular dinâmica temporal das regiões ativas pela média dos pixels na ROI sobre todas as imagens sem qualquer filtragem ou correção. </li></ol> Juntamente com o dextran Fura-2 enchemos os neurônios com o corante rodamina fixable dextran (&quot;mini ruby&quot;). Ambos os corantes têm o mesmo peso molecular, que devem ser co-localizados nos neurônios. Grande campo de imagem permite apenas a resolução espacial muito limitada, portanto recomenda-se a investigar as estruturas manchado após o experimento usando microscopia confocal. <ol><li> Após as medições fisiológicas, dissecar o cérebro e se fixar. </li><li> Enxágüe em PBS e desidratar stepwise em etanol e salicilato de metila em claro. </li><li> Microscopia confocal para incorporar cérebro em salicilato de metila. </li><li> Nós usamos um microscópio Leica TCS SP2. Comprimento de onda de excitação é 543nm. </li><li> Nós escanear o cérebro com um ar ou objetiva de imersão em óleo e comparar as estruturas coradas com os dados de imagem. </li></ol> Resultados representativos: Se a coloração e preparação foi bem-sucedida, o sinal na escala de cores falsas, por exemplo, em cogumelo corpo neurônios alfa lobo extrínsecos, poderia olhar como na figura 1. A resposta comportamental (PER) pode ser calculado como freqüência de pico durante a estimulação menos freqüência pico antes do estímulo (Figura 2). As estruturas manchadas podem ser investigadas em maior detalhe o uso de um microscópio confocal (Figura 3). <img src="/files/ftp_upload/1353/figure1_new.png" alt="Figura 1" /> Figura 1: Os sinais de cálcio Representante Imaging (estimulação com heptanal). A: região fotografada do cérebro das abelhas. B: image fluorescentes Raw como pode ser visto através de um mergulho X20 objetivo no comprimento de onda de excitação 380nm, o lobo-alfa do corpo cogumelo é descrito em roxo. Corante foi injetado no rim ventro-lateral dos lobos alfa-manchar neurônios extrínsecos. C: Imagem em cor falsa derivado do sinal de média, deltaF (340/380), durante o estímulo três segundo, alfa-lobo descritas em roxo, ROI selecionada delineadas em preto. D: dinâmica temporal do sinal de cálcio em resposta à estimulação odor, sinal média da região de interesse, como indicado no C. E: dinâmica temporal do sinal de cálcio em resposta à estimulação de sacarose da ipsi-e contra-lateral da antena, respectivamente para o ROI indicado em C. <img src="/files/ftp_upload/1353/figure2_new.png" alt="" /> Figura 2: resposta M17 Representante. Após o condicionamento olfativo a abelha estende a tromba quando o odor é apresentado sem recompensa. Linha vermelha indica o estímulo do odor. Detalhe: PER da abelha. <img src="/files/ftp_upload/1353/figure3_new.png" alt="" /> Figura 3: Representante da pilha de imagens. Após a experiência de imagem do cérebro foi dissecado. O neurônio da figura 1 é digitalizado usando um microscópio confocal com uma objetiva de imersão X20 petróleo, alfa-lobo descritas em roxo.

<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>

Nesta apresentação nós já passamos por todas as etapas para investigar sinais de cálcio vivo na abelha. Nós aplicamos essa técnica para os neurônios do corpo de cogumelo, mas de imagens pode ser feito em todos os neurônios para que uma técnica de coloração pode ser estabelecida. Temos nos concentrado em neurônios na via olfativa da abelha melífera. Os experimentos são realizados em uma câmara escura. A configuração é iluminado com uma luz de onda longa que se encontra fora da faixa d...

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

A in vivo e semi-vivo, em preparação para a imagem latente de cálcio tem sido desenvolvido em nosso laboratório por Joerges, Küttner Galizia e mais de dez anos atrás, para medir a atividade odor evocado no lobo antenal 1. A partir de então, ele tem sido constantemente refinados e aplicados a neuropiles diferentes no cérebro das abelhas. Aqui, descrevemos a preparação atualmente usado em laboratório para medir a atividade nos neurônios do corpo usando um cogumelo acoplado dextran cálcio sensíveis corante (Fura-2). Nós retrogradamente mancha neurônios corpo de cogumelos pela injeção de corante em seus axônios ou região soma. Nós nos concentramos em reduzir a invasão, para conseguir uma preparação em que ainda é possível treinar a abelha usando PER condicionado. Somos capazes de monitorar e quantificar a resposta comportamental gravando electro-myograms do músculo que controla o PER (M17) 2. Após a experiência fisiológica das estruturas imaged são investigados em maior detalhe por microscopia confocal para tratar a identidade dos neurônios.

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

Abelhas pode ser condicionado em um paradigma de aprendizado apetitivo olfativo (PER-condicionado). Usando odores como estímulos, estabelecemos um método em que o comportamento é gravado simultaneamente imagens de cálcio é usado para medir a atividade do odor evocado nos neurônios do corpo de cogumelo<em&gt; In vivo</em&gt;.

In vivo Ca 2 + - Imagem de Neurônios Corpo Mushroom durante o aprendizado olfativo no 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 visualizações.  Freie Universität Berlin. Abelhas pode ser condicionado em um paradigma de aprendizado apetitivo olfativo (PER-condicionado). Usando odores como estímulos, estabelecemos um método em que o comportamento é gravado simultaneamente imagens de cálcio é usado para medir a atividade do odor evocado nos neurônios do corpo de cogumelo In vivo.

Vídeo: 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.

Teste Comportamental olfativo no rato adulto

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

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 para Crônica In vivo</emGravação>: Fabricação Unidade

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 para Crônica In vivo</emGravação>: Assembléia Tetrodo

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.

Técnicas de imagem Ca 2 + Sinalização no esperma humano

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.

Análise Genética rápida de epitelial-mesenquimal sinalização durante a regeneração do cabelo

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.

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

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.

Obtenção de amostras com retardado, acelerado e invertida Envelhecimento no Modelo Bee Mel

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.

Métodos para comparar Nutrientes na beebread Feito por africanizadas e mel europeu abelhas e os efeitos sobre Títulos Hemolinfa Proteína

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.

Imagem local Ca<sup&gt; 2+</sup&gt; Sinais em células de mamíferos em cultura

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.

De alta resolução de imagem Time-lapse e análise automatizada de microtúbulos Dynamics em Viver umbilical humano Células Endoteliais da Veia

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