Este trabajo está financiado por la DFG.

Abeja Preparación y posterior relleno <ol><li> Captura recolectores de miel de abeja en la colmena y se enfría en hielo durante la inmovilización. </li><li> Montaje en Plexiglas las cámaras de los tres. Fijar los ojos y el tórax a las paredes de la cámara de grabación con cera de bajo punto de fusión duro. </li><li> Tire de capilares de vidrio como de uso general para la electrofisiología y la ruptura en la punta para obtener un diámetro de la punta de la ca. 10μm. Cobertura en la punta del capilar con pasta de tinte. Pasta de colorante consiste en una mezcla 10:01 de la Fura-2 dextrano y lisina puede arreglar tetrametilrodamina dextrano. </li><li> Para el procedimiento de tinción inmovilizar antenas con minucias o n eicosano. Extraer un pedazo de cutícula sobre el cerebro y las glándulas empuje y la tráquea hacia el lado para permitir el acceso al cuerpo de hongo. </li><li> Se inyecta en los capilares ya sea el soma o región axonal de las neuronas del cuerpo de setas. Esto se puede hacer manos libres o el uso de un micromanipulador. A continuación, restaurar piezas cutícula cápsula de la cabeza y aflojar las antenas. </li><li> Alimentar a las abejas con solución de sacarosa al 30% antes de guardarlos en una caja húmeda por lo menos durante cuatro horas o durante la noche a 20 ° C. </li></ol> En vivo de imágenes <ol><li> Evitar los movimientos de las abejas, por ejemplo con un pequeño pedazo de esponja que se aprieta contra el abdomen y se fija con un clip o una cinta en la cámara de grabación. </li><li> Para evitar el movimiento del cerebro como resultado del bombeo del esófago, el corte de una pequeña incisión en la cutícula por encima del labrum y sacar con cuidado el esófago y sus alrededores estructuras sólidas para ponerlo bajo tensión sin dañar el esófago 4. Cubierta de silicona de dos componentes. </li><li> Hacer agujeros con una aguja e insertar electrodos de alambre para la grabación de la M17. </li><li> Retire la pieza cutícula, la tráquea y las glándulas sobre el cerebro. Chupe heamolymph dentro de la cápsula con la cabeza un pedazo de papel. </li><li> Llene la cápsula de la cabeza con silicona de dos componentes. Es importante que el cerebro está completamente cubierto. </li><li> Abeja en lugar platina del microscopio y coloque una gota de agua en la superficie de la silicona para sumergir el objetivo de inmersión en el microscopio en la gota. Se centran en las neuronas de colores. </li></ol> Olor estimulación y registro de señales Olfatómetro: Nosotros usamos un ordenador controlado, personalizado dispositivo construido estimulación o &quot;olfatómetro&quot;, como se describe anteriormente por Galicia y Vetter 3. Los olores se diluyen en una corriente constante de aire dirigido hacia las antenas. Cualquier estímulo consiste en un impulso de olor 3s de aire saturado de olor a 0,2 ml. El protocolo de estimulación se puede ajustar en el TILLVision software de grabación. Imagen de microscopio y puesta en marcha: utilizamos un microscopio de fluorescencia Zeiss. Las imágenes se graban a 25 ° C con una tasa de muestreo de 5 Hz usando un dispositivo de Till-Fotónica de imagen montado en el microscopio. Señales de calcio se registran a través de un X60, Olympus 0,9 W inmersión objetivo con una cámara CCD Imago (640X480 pixeles, 4X agrupadas en el chip de hasta 160x120 píxeles). Fura-2 se excita con luz monocromática de 380 nm de longitud de onda 340 y para las mediciones radiométrica. La fluorescencia se detecta a través de un espejo dicroico 410 nm y un filtro de 440nm pase largo. Los parámetros para la grabación se encuentra en el software de grabación. Cada medida dura 10 segundos y el tiempo de exposición para las dos longitudes de onda se puede ajustar a la intensidad de la tinción en cada preparación. M17 grabación: El transportador muscular del labio (M17) se registra extracelularmente para monitorear las respuestas conductuales relacionados con el aprendizaje, es decir, por cada 5, 6. Se inyecta un hilo de cobre en el músculo cerca de la partes de la boca. Inyectar electrodo de tierra en el ojo. Los potenciales de los músculos son amplificadas con un encargo de la estructura pre-amplificador, digitalizadas y almacenadas en un ordenador. Inicio del estímulo se desencadena por el olfatómetro. Análisis de Datos <ol><li> Durante el experimento: Las grabaciones de la señal de calcio se introducen en un ordenador y se almacenan. El software de grabación TILLVision permite una inspección inicial de las señales, como el cálculo de la relación entre las longitudes de onda (340nm/380nm) y el cálculo de deltaF (sustracción de la línea de base). </li><li> Procesamiento de imágenes se realiza con la costumbre de los programas escritos en IDL. Calcular la relación de Ca 2 + señales de 340 nm y 380 nm mediciones para cada píxel. Determinar la fluorescencia de fondo (F) por un promedio de más cuadros antes que el estímulo y la resta de la señal radiométrica (deltaF). </li><li> Para definir la región de interés (ROI): Calcular media de fotogramas durante el estímulo y aplicar un filtro de paso bajo (3X3px). Convertir en escala de grises de la escala de colores falsos. Determinar la actividad neuronal en las estructuras de la actividad en forma de manchas en las imágenes en falso color y definir el ROI. </li><li> Calcular la dinámica temporal de las regiones activas de un promedio de los píxeles en el retorno de la inversión sobre todos los marcos, sin ningún tipo de filtro o corrección. </li></ol> Junto con el dextrano Fura-2 llenamos las neuronas con el colorante rodamina dextrano puede arreglar (&quot;mini rubí&quot;). Ambos colorantes tienen el mismo peso molecular, sino que debe ser colocado en las neuronas. Amplio campo de la imagen sólo permite una resolución espacial muy limitada, por lo tanto se recomienda para investigar las estructuras teñidas después del experimento mediante microscopía confocal de barrido. <ol><li> Después de las mediciones fisiológicas, diseccionar el cerebro y fijar. </li><li> Enjuague en PBS y se deshidratan por pasos en el etanol y el salicilato de metilo en claro. </li><li> Por microscopía confocal incrustar en el cerebro salicilato de metilo. </li><li> Nosotros usamos un microscopio Leica TCS SP2. Longitud de onda de excitación es 543nm. </li><li> Podemos escanear el cerebro con un objetivo de inmersión en aire o aceite y comparar las estructuras teñidas con los datos de imagen. </li></ol> Los resultados representativos: Si la coloración y la preparación se ha realizado correctamente, la señal en la escala de colores falsos, por ejemplo, en las neuronas de setas cuerpo alfa lóbulo extrínseca, se vería así en la figura 1. La respuesta conductual (PER) se puede calcular como la frecuencia de pico durante el estímulo menos frecuencia pico antes de que el estímulo (figura 2). Las estructuras teñidas pueden ser investigados con mayor detalle mediante un microscopio confocal (Figura 3). <img src="/files/ftp_upload/1353/figure1_new.png" alt="Figura 1" /> Figura 1: Representante de las señales de imágenes de calcio (estimulación con heptanal). R: Toma de imágenes de la región del cerebro de las abejas. B: imagen Raw fluorescentes como se ve a través de una inmersión X20 objetivo en la longitud de onda de excitación 380 nm, la alfa-lóbulo del cuerpo de setas se describe en la púrpura. Se inyectó en el borde ventro-lateral de los lóbulos alfa-para teñir las neuronas extrínsecas. C: Imagen en falso color deriva de la media de la señal, deltaF (340/380), en el estímulo de tres segundos, la alfa-lóbulo se indica en morado, seleccionados ROI delineados en negro. D: Dinámica temporal de la señal de calcio en respuesta a la estimulación olor, señal promedio de la región de interés, como se indica en el C. E: Dinámica temporal de la señal de calcio en respuesta a la estimulación de sacarosa de la ipsilateral y la antena contra-lateral, respectivamente, al retorno de la inversión indicada en C. <img src="/files/ftp_upload/1353/figure2_new.png" alt="" /> Figura 2: Representante M17 respuesta. Después del acondicionamiento olfativa de la abeja se extiende la probóscide, cuando el olor se presenta sin recompensa. La línea roja indica el estímulo del olor. Recuadro: PER de la abeja. <img src="/files/ftp_upload/1353/figure3_new.png" alt="" /> Figura 3: pila de imágenes Representante. Después del experimento de imágenes del cerebro se diseccionó. La neurona de la figura 1 se analiza con un microscopio confocal con un objetivo de inmersión en aceite de X20, la alfa-lóbulo se indica en morado.

<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>

En esta presentación se han seguido todos los pasos necesarios para investigar en las señales de calcio in vivo en la abeja melífera. Hemos aplicado esta técnica a las neuronas del cuerpo de hongos, pero las imágenes se puede realizar en todas las neuronas que puede ser una técnica de tinción establecida. Nos hemos centrado en las neuronas de la vía olfativa de la abeja de la miel. Los experimentos son realizados en un cuarto oscuro. La puesta en marcha se ilumina con una luz de onda l...

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

En vivo y semi-vivo en la preparación para obtener imágenes de calcio se ha desarrollado en nuestro laboratorio por Joerges, Kuttner y Galicia más de diez años, para medir la actividad olor evoca en el lóbulo antenal 1. A partir de entonces, ha sido constantemente perfeccionado y aplicado a neuropiles diferente en el cerebro de las abejas. Aquí se describe la preparación se utilizan actualmente en el laboratorio para medir la actividad en las neuronas del cuerpo de setas, junto con un dextrano colorante sensible al calcio (Fura-2). Nos retrógrada mancha de hongo neuronas del cuerpo mediante la inyección de contraste en sus axones o región soma. Nos centramos en la reducción de la invasión, para lograr una preparación en la que todavía es posible entrenar a las abejas con PER acondicionado. Somos capaces de controlar y cuantificar la respuesta de comportamiento mediante el registro electro-myograms del músculo que controla el PER (M17) 2. Después del experimento fisiológico las estructuras proyectadas se investigan con mayor detalle mediante microscopía confocal de barrido para hacer frente a la identidad de las neuronas.

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

Las abejas pueden ser acondicionadas en una paradigma de aprendizaje olfativo apetito (PER acondicionado). Utilizando los olores como estímulo, hemos establecido un método en el que el comportamiento se registra al mismo tiempo imágenes de calcio se utiliza para medir la actividad de olor evoca en las neuronas del cuerpo de setas<em&gt; In vivo</em&gt;.

En vivo Ca 2 + - Imágenes de las neuronas del cuerpo de hongos durante el aprendizaje olfativo en la abeja de la miel

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 vistas.  Freie Universität Berlin. Las abejas pueden ser acondicionadas en una paradigma de aprendizaje olfativo apetito (PER acondicionado). Utilizando los olores como estímulo, hemos establecido un método en el que el comportamiento se registra al mismo tiempo imágenes de calcio se utiliza para medir la actividad de olor evoca en las neuronas del cuerpo de setas 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.

Pruebas del comportamiento olfativo en el ratón adulto

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

Investigación

Biología

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-array de unidades de crónicos In vivo Grabación: Unidad de Fabricación

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-array de unidades de crónicos In vivo Grabación: Tetrodo Asamblea

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 Imagen Ca 2 + Señalización en el 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.

El análisis genético rápido de epitelio-mesenquimal de señalización durante la regeneración del cabello

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.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

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.

La obtención de especímenes con desacelerado, acelerado y Invertida Envejecimiento en la abeja de la miel Modelo

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.

Los métodos para la comparación de Nutrientes en jalea real Realizado por africanizadas y europeas Honey Bees y los efectos sobre títulos hemolinfa de proteínas

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.

Imagen Ca Local<sup&gt; 2 +</sup&gt; Las señales en las células de mamífero en cultivo

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.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

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