研究は、リンショーピン大学とオステルヨートランドの州議会によって資金を供給された。

1。背側経路を介して卵巣摘出<ol><li>酸素/亜酸化窒素30％/ 70％の混合物中にイソフルラン1.5％（誘導のための4％）の使用例については、動物を麻酔。直腸温度計に接続された加熱プレート上に腹臥位で動物を配置します。それは動物とプレート間の絶縁の薄い層を持つことが重要であり、温度計フィードバックシステムは、±0.5 37℃内で体温を保持する目の保護のためにゲルを適用し、術後の疼痛緩和のための動物の首の外側面に皮下に生理食塩水で希釈した5 mgのRimadyl / kg体重を管理します。 </li><li>腸骨稜からcephally 4×4センチ（マウスの3 * 3 cm）のエリアを剃る。徹底的に4×4 cmの開口手術のシートを持つ動物をカバーする前に、例Jodopaxために剃毛したエリアを洗います。 </li><li> 2〜3センチメートル正中切開を行い、ぶっきらぼうに、基礎となる筋膜から皮膚を解剖する。正中線の左右いずれかのCMが、別の切開のthroを作る筋膜ぐふっ。ぶっきらぼうに腹腔内に到達するまでなど、表面的に可能な限り、この筋膜の下に横方向に解剖する。 </li><li>ピンセットを使用して、腹腔内に卵巣を取り囲む脂肪組織をつかみ、静かに引き出します。卵巣を識別し、子宮角とこの構造体の近位血管0.5〜1センチメートルを連結。卵巣と連結された脂肪組織を切り取り、腹腔内に残っている組織のバックを入れた。反対側の筋膜内の別の切開を行い、手順を繰り返します。 </li><li>好ましくは、モノフィラメント縫合糸を使用して、傷を閉じて、麻酔から覚醒動物​​ましょう。手術後少なくとも2時間の間に加熱されたケージ（25-27°C）に動物を配置します。動物は、術後最初の数日間別々に収容する必要があります。それはケージは頻繁に回復期の間に清掃/交換されている極めて重要である。手術24時間後には、で希釈した別の5 mgのRimadyl / kg体重を皮下に動物を注入する生理食塩水。 </li></ol> 2。シラスティックカプセルを通じて17β-エストラジオールの皮下投与<ol><li>徹底的にゴマ油で17β-エストラジオールの所望量を混ぜる。ラットおよびマウスの生理的濃度のために、私たちはそれぞれ180μg/ mLとし、μg/ mLの18から36をお勧めします。プラセボカプセルは純粋なごま油で満たされている。 </li><li> 3センチメートル（マウス2 cm）のシラスティックチューブの長さおよび5mm（マウスの3 mm）をプラグインするための木製のアプリケーター棒の長さをカットします。鉗子を用いて、全体の長さを埋めるために17β-estradiol/sesame油溶液を注入する前に、シラスティックカプセルに1木製のプラグを絞る。静かに別の木栓（プラグがカプセルに永久に残ることになっている）で開放端にシラスティックカプセルをキャップ。注入前に一晩の残りの17β-エストラジオール溶液中でのカプセルをインキュベートします。 </li><li>カプセルの注入は、ラットの卵巣摘出で使用される麻酔中に実行することができます。マイルのためにCEは、しかし、卵巣摘出後14日間の回復期間が強く脆い皮膚から突き出ているカプセルのリスクを最小限にすることをお勧めします。 </li><li> 1.1のように手術の準備の後、または卵巣切開を閉じた後に、動物の首の背側面に2×2 cmの領域を剃ると洗ってください。 1cmの切開を行い、ぶっきらぼうにカプセルのための十分なスペースで、caudolaterally皮下ポケットを解剖。シラスティックカプセルを拭く（この時点まで、17β-エストラジオール溶液中でインキュベートされたことを）ゆっくりと、その後縫合糸で閉じられて切開を介してそれをインストールしてください。それは確認するために極めて重要であること、特に傷に近い領域で、できるだけ少ないようにカプセル株は皮膚、。カプセルの生産から注入まで、それは汚染が回避されることが重要です。 </li></ol> 3。ナットクリームヌテッラを経​​由して17β-エストラジオールの経口投与<ol><li>完全に溶解する17β-ヌテッラとごま油を混ぜた後ごま油でエストラジオール、と。私たちは、それぞれの日常の部分は17β-エストラジオール28μgの、5μLごま油ラットおよび各30グラムのマウス1.12μgの17β-エストラジオール、0.312μLごま油と60mgのNutellaのための体重1kgあたり1gのヌテッラが含まれていることをお勧めします。プラセボナットクリームは、17β-エストラジオールないことを除いて同じように用意されています。 </li><li>実験前に5日間ホルモンフリーのナットクリームを食べるために動物を訓練する。訓練の最初の3日間、ラットは最適自宅のケージ内のグループ（2-5動物に約0.5 gのヌテッラ）で訓練を受けるべきである。トレーニングの4番目と5番目の日に、動物実験の状況に似ている毎日の部分を受信する前に別のケージに入れられます。 </li><li>実験中に、動物はNutellaの部分は小型のセラミックタイルに提供された後、すべての給紙時に別々のケージに入れられます。訓練は、上記のOとして実行されている場合URの経験は、ラットやマウスの95％以上は、ナットクリームを受け入れて、一度完全に慣れて、動物のほとんどは数秒以内にそれを消費することです。 </li></ol> 4。代表的な結果現在の方法で再現することを目的と生理的血 ​​清17β-エストラジオール濃度は、ラットおよびマウスの4,6 5-35 pg / mLのために約5から140 pg / mLのです。皮下シラスティックカプセルは、上記の指示に従って、300グラム雌Sprague-Dawleyラットに投与した場合、徐々に約40 pg / mLの2日目からは10 pg / mLの35日に17β-エストラジオール血清濃度を減少させる生成します。しかし、すぐに投与した後、1000 pg / mLの周りの血中濃度のピークは4,5（ 図1A）8時間以内に生理的範囲に落ちて、期待できます。 25グラム雌C57BL / 6マウスに投与した36μgの17β-estradiol/mLと、上記のマウスのシラスティックカプセルは、血清濃塩酸をレンダリングゆっくりと約90 pg / mLの2日目から25 pg / mLの日35に減少entrations。これは（ 図1B）6 を検討されていないが、ラットで発見されたとして、これはおそらく同様のピークが付いています。 用量で経口Nutellaの方法は、300グラム雌Sprague-Dawleyラットに投与した場合、上記の10から70 pg / mLの（ 図2A）4との間の毎日の変動と17β-エストラジオールの生理的レベルをレンダリングします。上記の用量を用いたマウスでは、日常のレベルは約20〜120 pg / mLの（ 図2B）6との間で変動します。 <img alt="図1" src="/files/ftp_upload/4013/4013fig1.jpg" /> <st<html>ロン&gt;図1ラットの17β-エストラジオール濃度を（; n = 10で、後でポイントで24時間は、n = 15までの時間ポイントアップ）で提示する定型化されたグラフである。4,5およびマウス（B、N = 10）カプセル移植後6。シラスティックカプセルは、短い初期、1000 pg / mLのへの血清中濃度のピークが、100 pg / mLの下のレベルへの時間以内に減少を生成します。今後4-5週間では、カプセルは安定し、着実に減少するものの、血清中濃度を得ることができます。網掛け部分は、それぞれ、雌のラットとマウスにおける生理的17β-エストラジオール血清濃度に対応しています。視覚的にグラフで示されていませんであっても、個体間変動は（非常に低い平均27日から、ラットの183パーセントに、28からシラスティックカプセル法によるマウスの115％の変動係数をレンダリングし、17β-エストラジオール濃度で存在し濃度は頻繁に）変動の高い係数を生成します。 iles/ftp_upload/4013/4013fig2.jpg &quot;/&gt; 図2ラットの17β-エストラジオール濃度を提示する定型化されたグラフ（A; n = 15）で4およびマウス（B; n = 10）のNutellaの毎日のホルモン投与の結果として6。経口Nutellaのメソッドは、皮下または静脈内投与方法により製造されたとして高いしかし、しないことにより、これまでのところ、毎日のピークをレンダリングします。これは人間のホルモン補充療法の薬物動態を模倣し、経口治療は上記の例では、最初の96時間を提示している場合でも、必要なだけ長くするために進むことができます。網掛け部分は、それぞれ、雌のラットとマウスにおける生理的17β-エストラジオール血清濃度に対応しています。にもかかわらず、視覚的にグラフで示されていません、個体間変動は（非常に低い平均濃度Nutellaのメソッドを持つマウスではラットの40から132パーセントから、13から219％に変動係数をレンダリングし、17β-エストラジオール濃度で存在し多くの場合、高い生成変動係数）。

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著者らは、開示することは何もありません。

以前の研究を持っている上に示した管理モードでは、17β-エストラジオールの生理的血清濃度を生産の面で商業徐放ペレット、注射よりも優れて証明した。徐放ペレットは非常に高く、長時間の血清濃度のピークを生成しながら、注射剤は、極端な、毎日のホルモンの変動を引き起こし、動物4-6の繰り返しストレス処理を必要とします。適切な管理モードを選択することの重要性は、...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> Rimadyl、50 mg / mLの</td><td>ファイザー</td><td> 462986 </td><td>卵巣摘出後の術後鎮痛。 </td></tr><tr><td> Lubrithalアイジェル</td><td>レオ研究所</td><td></td><td>手術中の眼の保護。 </td></tr><tr><td> Jodopax獣医</td><td> Pharmaxim </td><td></td><td>外科消毒。 </td></tr><tr><td> Vicryl 4から0縫合糸</td><td>ジョンソン·アンド·ジョンソン</td><td></td><td>傷の縫合と卵巣結紮。 </td></tr><tr><td> 17β-エストラジオール、&gt; 98％ </td><td>シグマ</td><td> E8875-1G </td><td>シラスティックカプセルとNutellaのためのゴマ油で。 </td></tr><tr><td>ごま油</td><td>シグマ</td><td> S3547-1L </td><td> 17β-エストラジオールを溶解する。 </td></tr><tr><td>シラスティックチューブ、内径/外径：1.575/3.175ミリメートル</td><td>ダウコーニング</td><td> 508から008 </td><td>シラスティックカプセルの製造。 </td></tr><tr><td>木製のアプリケーター·スティック</td><td> Selefatrade </td><td> 12から0783 </td><td>シラスティックカプセルのキャップ。 </td></tr><tr><td>ヌテッラ</td><td>フェレロ</td><td></td><td> 17β-エストラジオール経口のための媒体。 </td></tr></tbody></table>

ovariectomy and 17&beta;-estradiol replacement in rats and mice: a visual demonstration

エストロゲンは、効果の非常に広いスペクトルを持つ女性の性ホルモンのファミリーである。ラットとマウスはエストロゲンの研究で使用されている場合、それらは一般的に17β-エストラジオール外因性の代わりに、急速にサイクリングホルモン産生を切除するために、卵巣摘出されています。ホルモンは生理的な血中濃度を得るために投与されるべきである方法に関するコンセンサスの欠如が、しかし、があります。 17β-エストラジオールレベル/管理方法が深く、実験結果は1-3に影響を与えるので、これは非常に重要です。我々は、皮下シラスティックカプセル単位および経口ナットクリームヌテッラは、市販の徐放ペレット（アメリカの会社の革新的な研究によって生成される）よりも優れているという発見は、17β-エストラジオール投与の異なるモードを特徴とする一連の研究であり、 17β-エストラジオール4-6の生理的血 ​​清濃度を生産の面で毎日注射。 ADVの中以前にブプレノルフィン投与7で使用されているナッツクリームの方法、のantagesは、エストロゲンの投与のために使用した場合には経口ホルモン補充療法に似ており、非侵襲的であるということです。皮下シラスティックカプセルは便利であり、最も安定した血清中濃度を生成します。このビデオの記事は、管理手順の重要な側面の議論に続いて卵巣摘出ラットおよびマウスのシラスティックカプセル及び経口ヌテッラによる17β-エストラジオールホルモン補充のステップバイステップのデモが含まれています。

Watch this Scientific Journal Video about Ovariectomy and 17Î²-estradiol Replacement in Rats and Mice: A Visual Demonstration at JoVE.com

Ovariectomy and 17&amp;#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

シラスティックカプセル及び経口ヌテッラによる卵巣摘出および17β-エストラジオールの後続の置換は、ラットとマウスで実証されています。

ラットとマウスにおける卵巣摘出および17β-エストラジオールの交換：ビジュアルデモ

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

ovariectomy-17-estradiol-replacement-rats-mice-visual

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Biology

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

56.7K ビュー.  Linköping University. シラスティックカプセル及び経口ヌテッラによる卵巣摘出および17β-エストラジオールの後続の置換は、ラットとマウスで実証されています。

ビデオ: Ovariectomy and 17&#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

ショウジョウバエの視覚と嗅覚を介したフライトコントロールを調査するために磁気テザーシステム

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

生物学

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2).
The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis.
The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3). 
The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

A demonstration of the bedside test for cutaneous allodynia and its clinical implications.

のデモンストレーション皮膚アロディニア慢性骨盤痛との関連で

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis.  In some women the severe episodes of cyclic pain change and the ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

マウスの錐体光受容体からの単一細胞サクション録音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

遺伝子誘導の運命のマッピングへの実践的アプローチ：マークとトラックの細胞へのビジュアルガイドインビボでの</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

マウスとラットの拘束マニュアルと共通の複合投与経路

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

診断剖検及びラット及びマウスで選択された組織とサンプル集

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

ショウジョウバエにおける温度嗜好挙動とその概日リズムの設計と解析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

生理学ラボデモンストレーション：ラットにおける糸球体濾過率

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

細胞特異的にLINC複合体を破壊するマウスモデルの検証

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

生体内でRb タンパク質 sumo 化ひと細胞の検出と解析

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...