Bu çalışma DFG tarafından finanse edilmektedir.

Arı Hazırlık ve Geri dolgu <ol><li> Immobilizasyon için buz üzerinde kovanı ve soğuk bal arısı yem yakalayın. </li><li> Pleksiglas odaları 3 kayıt Dağı. Gözleri ve göğüs düşük erime noktası sabit balmumu ile kayıt odasının duvarları sabitleyin. </li><li> Elektrofizyoloji için yaygın olarak kullanılan cam kılcal damarların çekin ve yaklaşık bir ucu çapını elde etmek için ucunda kırmak. 10μm. Kapak boya macun ile kılcal ucu. Boya yapıştırın Fura-2 dekstran ve lizin tamir edilebilir tetramethylrhodamine dekstran 10:01 karışımından oluşur. </li><li> Boyama işlemi için önemsiz ayrıntılar veya n-eicosane antenler hareketsiz. Beyin yukarıda manikür bir parça çıkarın ve mantar vücut erişimine izin vermek yan bezleri ve trakea itin. </li><li> Soma veya mantar vücut nöronların aksonal bölge içine kılcal damarların enjekte edilir. Bu serbest elle yapılan veya mikro manipülatör kullanarak olabilir. Sonra baş kapsül kütikula parça geri ve antenler gevşetin. </li><li> % 30 sukroz solüsyonu ile arılar Yem nemlendirilmiş bir durumda onları en az dört saat süreyle saklamadan önce ya da gece boyunca 20 ° C </li></ol> In vivo Görüntüleme <ol><li> Karın karşı bastırdı ve kayıt odasına bir klip veya bant ile sabit küçük bir parça sünger ile arı, örneğin hareketleri engelleyin. </li><li> Labrum yukarıdaki manikür, yemek borusu pompa kaynaklanan beyin hareketi önlemek için, içine küçük bir kesi kesme ve yemek borusu dikkatlice dışarı çekin ve çevresindeki sağlam yapılar, yemek borusuna 4 zarar vermeden gerilim altında koymak. Iki bileşenli silikon ile kaplayın. </li><li> Bir iğne ile delik açın ve M17 kayıt için tel elektrotlar eklemek. </li><li> Kütikula parça, trakea ve beyin üzerinde bezleri çıkarın. Bir kağıt parçası ile baş kapsül içinde heamolymph Suck. </li><li> Iki bileşenli silikon ile baş kapsül doldurun. Beyin üstünün tamamen kapalı olduğundan çok önemlidir. </li><li> Mikroskop sahne arı ve damlacık içine mikroskop daldırma amacı batırmayın silikon yüzey üzerinde bir damla su yerleştirin. Lekeli nöronlar odaklanın. </li></ol> Koku uyarılması ve kayıt sinyallerin Olfactometer: Biz bir bilgisayar kontrollü, özel olarak inşa stimülasyon cihazı veya Galizia ve Vetter 3 tarafından daha önce açıklandığı gibi &quot;olfactometer&quot; kullanın. Kokular antenler yönelik sürekli bir hava akışı içine seyreltilir. Herhangi bir uyaran bir 3s koku darbe 0.2ml koku doymuş hava oluşur. Stimülasyon protokolü kayıt yazılımı TILLVision ayarlanabilir. Mikroskop ve Görüntüleme set-up: Biz Zeiss bir floresan mikroskop kullanıyoruz. Görüntüler 25 ° C mikroskop üzerine monte edilmiş bir KADAR-Fotonik görüntüleme cihazı kullanarak 5 Hz&#39;lik bir örnekleme hızı ile kaydedilir. Kalsiyum sinyalleri bir X60, 0.9 W Olympus Imago CCD kamera (640x480 piksel, 160x120 piksel çip üzerinde binned 4X) daldırma amacı ile kayıt altına alınmaktadır. Fura-2 monokromatik 340 oranlı metrik ölçümleri için ışık ve 380nm dalga boyu ile heyecanlı. Floresan 410 dikroik ayna ve 440nm uzun geçiren filtre ile tespit edilir. Kayıt yazılımı kayıt için parametreler ayarlanır. Her ölçüm iki dalga boylarında ve pozlama süresi 10s her hazırlık boyama yoğunluğu ayarlanabilir sürer. M17 kayıt: labium (M17) iletki kas öğrenme ile ilgili davranışsal tepkilerin izlemek için ekstraselüler kaydedilir BAŞINA 5, 6, yani. Bakır tel ağız parçaları yakın kas içine enjekte edilir. Toprak elektrot göz içine enjekte edilir. Kas potansiyelleri, özel bir yapı pre-amplifikatörü ile amplifiye sayısallaştırılmış ve bir bilgisayarda depolanır. Stimulus başlangıçlı olfactometer tarafından tetiklenir. Veri Analizi <ol><li> Deney sırasında bir bilgisayar içine kalsiyum sinyal kayıtları beslenir ve saklanır. Kayıt yazılımı TILLVision dalga boylarında (340nm/380nm) ve deltaF (bazal çıkarma) hesaplanması arasındaki oranın hesaplanması gibi, sinyallerin bir ilk denetim sağlar. </li><li> Görüntü işleme IDL özel yazılmış programlar ile yapılır. Ca 2 + 340nm gelen sinyalleri ve her piksel için 380nm ölçüm oranını hesaplayın. Uyaran önce kare ortalama eşiğe (F) belirlemek ve oranlı metrik sinyali (deltaF) çıkarmak. </li><li> Faiz (ROI) Bölge tanımlamak için: uyaran sırasında kare ortalama ve bir alçak geçiren filtre (3X3px) uygulamak hesaplayın. Yanlış renk skalası gri ölçekli dönüştürün. Sahte renkli görüntüler faaliyet lekeler olarak nöronal yapılardaki aktiviteyi belirleyin ve yatırım getirisi olarak tanımlamak. </li><li> Herhangi bir filtreleme veya düzeltme olmadan tüm kareleri üzerinde yatırım getirisi piksellerin ortalamasını alarak aktif olan bölgelerin geçici dinamikleri hesaplayın. </li></ol> Fura-2 dekstran ile birlikte biz tamir edilebilir boya rodamin dekstran (&quot;mini yakut&quot;) ile nöronların doldurun. Her iki boyalar aynı moleküler ağırlığa sahip nöronlar birlikte yer almalıdır. Geniş açılı görüntüleme, sadece çok sınırlı uzaysal çözünürlüğü sağlar, bu nedenle konfokal tarama mikroskobu kullanılarak deney sonra lekeli yapılarını araştırmak için tavsiye edilir. <ol><li> Fizyolojik ölçümler sonrası, beyin incelemek ve sabitleşmek. </li><li> PBS ve kademeli dihidrat etanol ve metil salycilate net durulayın. </li><li> Konfokal mikroskobi için metil salycilate beyin gömün. </li><li> Biz Leica TCS SP2 mikroskop kullanıyoruz. Dalgaboyu 543nm. </li><li> Biz bir hava veya yağ daldırma amacı ile beyin tarama ve lekeli yapıların görüntüleme verileri ile karşılaştırmak. </li></ol> Temsilcisi Sonuçlar: Boyama ve hazırlık başarılı olursa, yanlış renk skalası sinyal, mantar vücut alfa lob ekstrensek nöronlar Örneğin, Şekil 1 gibi görünebilir. (PER) ve davranışsal tepki, uyarana (Şekil 2) önce uyaran eksi başak frekans sırasında başak frekans olarak hesaplanabilir. Lekeli yapılar, konfokal mikroskop kullanılarak (Şekil 3) daha ayrıntılı araştırılması olabilir. <img src="/files/ftp_upload/1353/figure1_new.png" alt="Şekil 1" /> Şekil 1: Kalsiyum Temsilcisi Görüntüleme sinyalleri (heptanal ile stimülasyon). A: arı beyni görüntülenmistir bölge. B: 380nm dalgaboyu X20 daldırma amacı ile görüldüğü gibi ham floresan görüntü, mantar vücudun alfa-lob mor özetlenen. Boya ekstrensek nöronlar leke alfa-lobların ventro-lateral kenarı içine enjekte edildi. C: üç ikinci uyaran sırasında ortalama sinyali deltaF (340/380), türetilmiş Yanlış renkli görüntü; mor belirtilen alfa-lob, seçilen ROI siyah sıraladı. D: koku stimülasyon, C. E belirtildiği gibi ilgi bölgeden ortalama sinyal yanıt Geçici kalsiyum sinyal dinamikleri: ROI sırasıyla sukroz uyarılması IPSI ve kontra-lateral anten yanıt kalsiyum sinyal Geçici dinamikleri, C. içinde <img src="/files/ftp_upload/1353/figure2_new.png" alt="" /> Şekil 2: M17 yanıt Temsilcisi. Koku alma Klima sonra arı koku ödül olmadan burnumun uzanır. Kırmızı çizgi, koku uyaran gösterir. Ankastre: balarısı, PER. <img src="/files/ftp_upload/1353/figure3_new.png" alt="" /> Şekil 3: Temsilci görüntü yığını. Sonra beyin görüntüleme deney disseke edildi. Şekil 1 nöron mor belirtilen bir X20 immersiyon yağı amacı, alfa-lob ile konfokal bir mikroskop kullanılarak taranır.

<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>

Bu sunumda, balarısı in vivo kalsiyum sinyallerini araştırmak için tüm adımları aracılığıyla gitti . Biz mantar vücut nöronlar için bu tekniği uygulanabilir, ancak bir boyama tekniği kurulacak olan bütün nöronlar üzerinde görüntüleme yapılabilir. Biz bal arısı koku yol nöronların odaklanmıştır. Deneyleri bir karanlık oda yapılır. Kurmak arı optik algılama aralığı yalan ve boyalar heyecanlandırmak değil uzun dalga boyundaki ışık ile aydınlatılır. Örneğin silikon hava kabarcıkları, herhangi bir flüoresan bozulmaları önlemek için önemlidir. Bu sistemin en önemli avantajlarından biri, mekansal ve zamansal çözünürlük ile nöron faaliyetleri hakkında bilgi örnek olasılığıdır. Örneğin nöron öğrenme ile ilgili plastisite araştırılmalıdır Aynı zamanda, biz arı ilgili davranışlarını gözlemlemek olabilir. Ancak, mekansal ve zamansal çözünürlüğü sınırlıdır. In vivo kalsiyum görüntüleme kullanarak, deneysel süresi sınırlıdır. Ekstrasellüler elektrofizyolojik yöntemlerle, kayıtları, birkaç saat veya gün sürebilir, iken, deneyler, yaklaşık 90 dakika ile sınırlıdır. Biz bir 5Hz örnekleme oranı, her bir ölçüm 10 saniye süren, 20ms ve 380nm maruz kalıcı 5ms, 340nm maruz kalma, sırasıyla iki dalga boylarında uyarma ile 85 ölçüm gerçekleştirdiğinizde sinyaller yine de oldukça iyi. Daha sonra bir sorun haline gelebilir beyazlatma boya.

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

Antennal lob 1 in vivo ve yarı-Kalsiyum görüntüleme için in vivo hazırlık Joerges, Kuttner ve Galizia laboratuarımızda geliştirilmiştir üzerinden on yıl önce, koku uyarılmış aktivitesini ölçmek için. O andan itibaren, sürekli olarak rafine edilmiş ve arı beyni farklı neuropiles uygulanır. Burada henüz bir dekstran birleştiğinde kalsiyum duyarlı boya (Fura-2) kullanarak mantar vücut nöron aktivitesini ölçmek için laboratuarda kullanılan hazırlık açıklar. Biz retrograd mantar vücut nöronların aksonları ya da soma bölge içine boya enjekte edilerek leke. Biz klima BAŞINA kullanarak arı yetiştirmek için hala mümkün olduğu bir hazırlık elde etmek için, invazivlik azaltarak odaklanmak. PER (M17) 2 kontrol eden kas elektro-myograms kayıt davranışsal tepki ölçmek ve izlemek mümkün. Fizyolojik deney sonra görüntülü yapılar nöronların kimlik adresine konfokal tarama mikroskobu kullanılarak daha ayrıntılı olarak incelenmiştir.

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

Arılar iştahlı bir koku alma öğrenme paradigması (PER-condition) klimalı olabilir. Uyaranlara gibi kokular kullanarak, aynı anda Kalsiyum Görüntüleme koku uyarılmış mantar vücut nöron aktivitesini ölçmek için kullanılır kaydedilir hangi davranış bir yöntem kurdu<em&gt; In vivo</em&gt;.

In vivo Ca 2 + - Bal Arısı Koku Öğrenme sırasında Mantar Vücut Nöronlar Görüntüleme

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

Universidad San Sebastian

Research

JoVE Journal

Biology

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

13.9K Görüntüleme.  Freie Universität Berlin. Arılar iştahlı bir koku alma öğrenme paradigması (PER-condition) klimalı olabilir. Uyaranlara gibi kokular kullanarak, aynı anda Kalsiyum Görüntüleme koku uyarılmış mantar vücut nöron aktivitesini ölçmek için kullanılır kaydedilir hangi davranış bir yöntem kurdu 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.

Yetişkin Fare Olfaktör Davranış Testi

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

Biyoloji

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-sürücü Kronik Array In vivo Kayıt: Tetrode Meclisi

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.

Görüntüleme Ca Teknikleri 2 + İnsan Sperm sinyal

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.

Saç Rejenerasyon sırasında epitelyal-mezenkimal Sinyal Rapid Genetik Analizi

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.

Apoptotik hücresi Temizliği görüntülenmesi sırasında Canlı<em&gt; Drosophila</em&gt; Embriyo

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.

, Yavaşlatıldı Hızlandırılmış ve Ters Honey Bee Model Yaşlanma ile Örneklerin Alınması

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.

Africanized ve Avrupa Bal Arısı Made by beebread besin tuzu karşılaştırılması yöntemleri ve Hemolenf Protein Titrelerin Etkileri

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.

Görüntüleme Yerel Ca<sup&gt; 2+</sup&gt; Kültürlenmiş memeli hücrelerinde sinyaller

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.

Yüksek çözünürlüklü Time-lapse Görüntüleme ve Yaşayan İnsan Umbilikal Ven Endotelyal Hücrelerinde Mikrotubul Dinamiği Otomatik Analizi

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

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

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

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

Kalp Fonksiyon Çalışmaları Diseksiyon ve Bal Arısı Dorsal Vessel Gözlem

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