この作業はDFGによって運営されている。

蜂の準備とバックフィル<ol><li>固定化のため氷の上でハイブとチルでミツバチの飼料収穫機をキャッチ。 </li><li>プレキシグラス録音室3でマウントします。低融点のハードワックスとレコーディング室の壁に目と胸部を修正。 </li><li>一般的に電気生理学のために使用されるガラスキャピラリーを引いて、CAの先端径を得るために先端に壊れる。 10μmの。染料ペーストとキャピラリーの先端をカバーしています。染料ペーストは、フラ-2デキストランおよびリジン修正可能なテトラメチルローダミンデキストランの10:1混合物で構成されています。 </li><li>染色手順のために特徴点またはn -エイコサンとアンテナを固定する。脳上記のキューティクルの一部を削除し、キノコ体へのアクセスを許可する側に腺と気管を押してください。 </li><li>ソーマやキノコ体の神経細胞の軸索領域のいずれかに毛細血管を注入する。これは、フリーハンドを行ったり、マイクロマニピュレータを使用することができます。その後、頭部カプセルでキューティクル部分を復元し、アンテナを緩めます。 </li><li> 20℃で少なくとも4時間または一晩加湿場合にそれらを格納する前に、30％ショ糖溶液と蜂を養う℃に</li></ol>生体内撮像<ol><li>腹部に押し付けると、録音室にクリップやテープで固定されているスポンジの小片で蜂などの動きを妨げる。 </li><li>食道の汲み上げに起因する脳の動きを防ぐために、唇上記のキューティクルに小さな切開を切断し、食道の4を損なうことなく、緊張下に置くために慎重に食道とその周囲の固体構造を引き出します。二成分シリコーンをカバー。 </li><li>針で穴を確認し、M17の記録のためのワイヤ電極を挿入する。 </li><li>キューティクル部分、気管、脳、上記の腺を削除します。一枚の紙で頭部カプセル内heamolymphを吸う。 </li><li>二成分シリコーンを頭のカプセルを埋める。脳が完全に覆われていることが重要です。 </li><li>場所を顕微鏡ステージ上に蜂と液滴に顕微鏡のディップの目標を浸すために、シリコンの表面に水滴を置く。ステンドニューロンに焦点を当てる。 </li></ol>信号の匂いの刺激と記録 嗅覚：我々は、コンピュータ制御された、カスタム構築された刺激装置またはGaliziaとフェッター3で前述したように&quot;嗅覚&quot;を使用。悪臭は、アンテナに向け一定の空気流中に希釈されています。あらゆる刺激は、0.2ミリリットル臭い飽和空気の3S臭のパルスで構成されています。刺激のプロトコルは、録音ソフトウェアTILLVisionに設定することができます。 顕微鏡とイメージングセットアップ：我々はツァイス蛍光顕微鏡を使用する。画像は25 °顕微鏡に搭載されたTILLフォトニクスイメージング装置を用いて5 Hzのサンプリングレートを持つCで記録されている。カルシウムの信号はX60、0.9 Wオリンパス成虫CCDカメラ（640x480ピクセル、160x120ピクセルのチップ上でビニング4X）とディップ目的によって記録されます。フラ-2は、レシオメトリックな測定のための340および380nmの波長の単色光で励起される。蛍光は、410nmのダイクロイックミラーと440nmロングパスフィルタを介して検出されます。記録のためのパラメータは、レコーディングソフトウェアで設定されます。各測定には2つの波長に対して10秒と露光時間をそれぞれ準備の染色強度に調整することができます続きます。 M17の記録：唇（M17）の伸筋が学習に関連する行動反応を監視するために細胞外記録され、PER 5、6、すなわち。口の部分に近い筋肉に銅線を注入する。目に接地電極を注入する。筋肉の電位は、カスタムビルドプリアンプで増幅デジタル化してコンピュータに保存されます。刺激の発症は、嗅覚によってトリガされます。 データ解析<ol><li>実験中：カルシウム信号の録音は、コンピュータに供給され、格納されています。レコーディングソフトウェアのTILLVisionは波長（340nm/380nm）とdeltaF（ベースラインの減算）の計算の間の比率の計算のように、信号の最初の検査が可能になります。 </li><li>画像処理は、IDLでカスタム作成されたプログラムで実行されます。のCa 2の比を計算する+各ピクセルの340nmと380nmの測定値からの信号を。刺激前のフレームにわたって平均化することにより、バックグラウンド蛍光（F）を決定し、レシオメトリック信号（deltaF）から差し引きます。 </li><li>対象領域（ROI）を定義するには：刺激中にフレームの平均とローパスフィルタ（3X3px）を適用する計算。偽色のスケールにグレースケールに変換します。偽色の画像でのアクティビティのスポットとしてニューロン構造の活動を決定し、ROIとして定義する。 </li><li>いかなるフィルタリングまたは修正することなくすべてのフレームにわたってROI内のピクセルを平均化することにより活性領域の時間的なダイナミクスを計算する。 </li></ol>一緒にフラ-2デキストランで我々は解決不能色素ローダミンデキストラン（&quot;ミニルビー&quot;）でニューロンを埋める。両方の色素は同じ分子量を有し、それらは神経細胞に共存する必要があります。広視野イメージングは​​非常に限られた空間分解能を可能にする、従ってそれは、走査型共焦点顕微鏡を用いて実験した後に染色した構造を調べることをお勧めします。 <ol><li>生理学的測定の後、脳を解剖し、凝視する。 </li><li> PBSおよびエタノールとメチルsalycilateで明確に段階的に脱水にすすいでください。 </li><li>共焦点顕微鏡のためのメチルsalycilateで脳を埋め込みます。 </li><li>私たちは、ライカTCS SP2顕微鏡を使用してください。励起波長は543nmです。 </li><li>我々は、空気や油浸対物レンズで脳をスキャンし、画像データで染色した構造を比較する。 </li></ol>代表的な結果： 染色と準備が成功した場合、偽色のスケールに信号は、キノコ体α葉外因性ニューロンで、例えば、図1のようになります。 行動反応は（PER）刺激（図2）の前に刺激マイナススパイク​​の周波数の間にスパイク頻度として計算することができます。 ステンド構造は、共焦点顕微鏡（図3）を用いてより詳細に検討されることがあります。 <img src="/files/ftp_upload/1353/figure1_new.png" alt="図1" /> 図1：代表的なカルシウムイメージングの信号（ヘプタナール刺激）。 ：蜂の脳の画像化された地域。 B：380nm励起波長におけるX20ディップ客観を通して見た、キノコ体のα-葉は紫色で概説されている生の蛍光画像。染料は、外因性神経細胞を染色するためにα-ローブの腹外側縁に注入した。 C：3つの第2の刺激の間の平均信号、deltaF（380分の340）に由来する偽色の画像、α-葉紫、黒に記載されている選択されたROIに記載されている。 D：匂いの刺激、C. Eに示すように関心領域から平均的な信号に応答してカルシウムシグナルの時空間ダイナミクス：ROIにそれぞれショ糖の刺激IPSI -と対側のアンテナへの応答におけるカルシウムシグナルの時空間ダイナミクスが示されたC.で<img src="/files/ftp_upload/1353/figure2_new.png" alt="" /> 図2：代表M17応答。嗅覚コンディショニング後蜂は匂いが報酬なしで提示される口吻を拡張します。赤い線は臭いの刺激を示している。挿入図：ミツバチのPER。 <img src="/files/ftp_upload/1353/figure3_new.png" alt="" /> 図3：代表画像スタック。イメージング実験後に脳を解剖した。図1からニューロンは紫で概説X20の油浸対物レンズ、α-ローブに共焦点顕微鏡を用いてスキャンされます。

<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>

このプレゼンテーションでは、ミツバチの in vivoカルシウムシグナルに調査するためにすべてのステップを経ています。我々は、キノコ体の神経細胞にこの手法を適用し、が、画像は、染色技術を確立することができるため、すべてのニューロンに行われることがあります。我々は、ミツバチの嗅覚路における神経細胞に焦点を当てている。実験はすべて暗室で行われる。セッ?...

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

生体とセミのカルシウムイメージングのためのin vivoでの準備でカットナーとGaliziaは、触角葉1の臭気誘発活性を測定するために、十年以上前に、Joergesによって私たちの研究室で開発されています。その時から、それが継続的に洗練されており、ミツバチの脳の異なるneuropilesに適用される。ここで、我々は現在、デキストラン結合のカルシウム感受性色素（フラ-2）を使用して、キノコ体の神経細胞で活性を測定するために実験室で使用されている準備について説明します。我々は、逆行軸索や細胞体の領域に色素を注入することによりキノコ体の神経細胞を染色。我々は、それがコンディショニングのPERを使用してハチを訓練することは可能である準備を達成するために、侵襲性を減らすことに焦点を当てる。我々は、PER（M17）2を制御する筋肉からの電気myogramsを記録することによって、行動反応を監視し、定量化することができます。 生理学的実験の後に画像形成された構造は、ニューロンの識別に対処するための走査型共焦点顕微鏡を用いてより詳細に検討されています。

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

ミツバチは、食欲の嗅覚学習のパラダイム（PER -コンディショニング）で条件付けることができます。刺激として匂いを使用して、我々は、同時にカルシウムイメージングがキノコ体ニューロンの匂い誘発活性を測定するために使用されている間に記録されている動作にする方法を確立<em&gt; in vivoで</em&gt;。

ハニービーの嗅覚学習中のキノコ体の神経細胞のイメージング- 生体内のCa 2 + の

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 ビュー.  Freie Universität Berlin. ミツバチは、食欲の嗅覚学習のパラダイム（PER -コンディショニング）で条件付けることができます。刺激として匂いを使用して、我々は、同時にカルシウムイメージングがキノコ体ニューロンの匂い誘発活性を測定するために使用されている間に記録されている動作にする方法を確立 in vivoで。

ビデオ: 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.

成体マウスでの嗅覚行動試験

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

生物学

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.

慢性のためのマイクロドライブアレイ in vivoで録音：ドラ​​イブの作製

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.

慢性のためのマイクロドライブアレイ in vivoで録音：四極管アセンブリ

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.

イメージングカルシウムのためのテクニック 2 +人間の精子でシグナリング

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.

髪の再生の間に上皮間葉シグナル伝達の迅速遺伝子解析

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.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

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.

ハニービーモデルの老化、減速し加速し、逆に試験片を得た

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と欧州のハニーミツバチによって作らBeebreadで栄養素を比較するための方法及び体液タンパク質力価への影響

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.

イメージングローカルCA<sup&gt; 2+</sup&gt;培養哺乳動物細胞内シグナル

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.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

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