FONCICYT 000000000091984; Jeansson、セーデルストレームKönigska sjukhemmet、マーサLundqvistさんとシグルドOCHエルザの基礎この作品は、スウェーデンの医学研究評議会（K2009 - 75SX - 21028〜01 - 3、K2008 - 61X - 20700〜01 - 3）によって賄われていたGoljesミンヌ、および医学SLS - 95151のスウェーデンの社会。教授ジーンD.ブロック、UCLAは、感謝して原稿上で貴重なコメントで知られています。我々はビデオ顕微鏡を提供するための博士マイケルAndäng博士とヘレナJohard HCNチャネルブロッカーのため、教授とアブデルエルManiraに感謝する。 倫理的配慮： 動物のすべての実験はカロリンスカ研究所と&quot;ストックホルムのノーラDjurförsöksetiska Nämnd&quot;で定められたガイドラインおよび規制に準拠して行った。全ての動物実験は、動物へのあらゆる可能なストレスや不快感を最小限に抑える目的で行われます。

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ルシフェラーゼレポーターの技術を持つメリットとデメリット昼間の勉強するために多くの異なる時点（サンプリング周波数に応じて、通常2-4時間の低時間分解能を与える）で組織採取を必要とするin situハイブリダイゼーション及びウエスタンブロットで 、このようなRT - PCRなどのex vivoでの方法とは対照的に、遺伝子と蛋白質の表現の変化、ルシフェラーゼレポーター技術は、高解像度（1-10分）と同じ準備で何日も概日振動の研究をすることができます。したがって、使用する動物の数は最小限に抑えられ、リズムの位相と周期への影響の詳細な研究は、通常、低時間分解能と、従来のサンプリング技術を利用可能ではない、実現可能である。しかし、リズムの相対的な振幅の測定が可能であるが、それはレポーター技術は定量的でないため、転写された遺伝子または翻訳されたタンパク質の量を測定するために使用することができないことが強調されるべきである。 組織の記録は、器官SCNの文化は成人の脳の長期的な（週）薬理学的操作を可能に開始し、直ちに培養、直接生体内光誘起位相シフトなどで 、 生体のストレスでの分子の影響を研究するための大きな利点の後に分析することができます無傷の成熟したシナプスのネットワークとルシフェラーゼレポーターの技術を含む組織は、何週間も安定した記録が可能になります。したがって、組織が健全な文化を得るために新生児または出生後早期にする必要はありません、と急性スライス慢性の薬理学的治療とは対照的に行うことができます。エピジェネティックな変化が検討される場合はないゲノムへのDNAの取り込みにつながるか細胞培養におけるプラスミド - レポータートランスフェクション、とは対照的に、トランスジェニックルシフェラーゼ - 動物モデル（だけでなく、レンティ-ウイルスのトランスフェクション）が良好である。それはこの文脈でも発光イメージングは、レコーディングにも概日リズムを研究の主要な研究室によって利用される単一のSCNのニューロン（〜60から10ミクロン）や他の細胞型、許されるように、高感度CCDカメラ11で、今日可能であることが述べられるべきである。最後に、いくつかの概日研究者は、一般的に時計遺伝子/蛋白質の振動16-19を勉強するために、そのような緑色蛍光タンパク質として、他のレポーターを用いた分子の発現のモニタリングを使用してください。 スライス厚の側面必要に応じて、ここで推奨SCNのスライス（200〜300ミクロン）の厚さが減少または増加させることができた。組織の文化の中で数日後に膜に平坦化するが、しかし、、それは組織7の実行可能性を維持するために最初にカットスライスの500μmの厚さを超えることは推奨されません。が100μmよりも薄いスライスは取り扱いが困難機械的および実際的な理由からです。 SCNの組織が不均質であるため、SCN（例えば、&quot;シェル&quot;と&quot;コア&quot;、と背比べ腹側の領域）内の異なる領域が異なる位相で振動するので、スライスの厚さは、ルシフェラーゼの出力信号の位相に影響を及ぼす可能性がありますフェーズの後に20と差動再同期は、21をシフト。より多くの組織は、光子の大きい数を発するので、スライスの厚さも光子数のベースラインに影響を与えます。分子振動の振幅は、このように間接的に植の大きさによって影響を受ける可能性があります。これらの理由から、制御と処理培養物は、常に同じ大きさ、厚さであることと同じ位相で、同じ振幅で発振するために、SCNの同じ領域を含む必要があります。 two一方的な核は、その一方で、ビブラトームカットが順番に小脳と二つの半球の間に切断分離が垂直であることに依存している水平方向と（角度がない限り、お互いにと同様の振幅と位相で振動するテーブルの表面に）。 SCNの培養を行う研究者は、SCNの文化は互いに位相で発振しないことが発生する可能性があります。練習とスライシング技術の標準化、スライスは常にSCNの同じrostro -尾レベルでカットされている、すなわち、結果が改善されます。 重要なステップ<ol><li>酸素供給は重要です。 脳は酸素22の多くを必要とするため、スライシング手続きは迅速である必要がある（3.1から3.10に近い、より良い、それが組織を健康に保つためにある〜5-6分です）。 </li><li> SCNは、温度変化や培地交換に敏感です。 SCNは、温度に敏感であり、大きな温度変化は、位相ペースメーカーをシフトし、実験的なアーティファクト23を引き起こす可能性があります。培地やソリューションは、培養中の数日後に薬を適用するなどの継続的な実験、、培地またはsolutio中に交換または追加される場合nは、さらに前に36〜37 ° C（チャンバー内と同じ温度）に事前に温める必要があります。培地交換を伴う薬理学的実験では、それは常に事前にエアコンの細胞培養培地と連携することが重要です。さらに/新鮮な、無条件の培地でアウト洗浄は、潜在的に分子のSCNのリズム24だけでなく、他の細胞型の培養25を位相シフトすることができます。 </li><li>培地組成物。 正しいpH及び浸透圧の文化組織をすることが重要です。空気緩衝培地はpHが6.8から8.2の範囲で最適なバッファ容量を持ち、細胞の呼吸の結果として発生する可能性のあるpHの変化をバッファリングするためにも適切であるHEPESが大量に含まれています。 HEPESは、一方、として培地中に少量に含まれている炭酸水素ナトリウム、と比較して温度変化に対して敏感です。重炭酸ナトリウムは、CO 2雰囲気下でこのように適切な低いpH（5.1〜7.1）範囲26内のより大きな緩衝能力を持っています。しかし、空気緩衝培地（DMEM 2902）に重炭酸ナトリウムの量を増加が大幅に浸透圧を増加させると同時に培地、アミノ酸、ビタミンおよびイオンの不正確な組成のターン結果に含めます。希釈することなく追加することができます。 培地を調製するためのアドバイス： <ol><li>媒体とを混合する際、正確なと注意してください。 </li><li>だけ新鮮か新鮮解凍したストック溶液を使用してください。 </li><li>非常に高い浸透圧の場合には、希釈する価値はありません。以上の5から6パーセントに希釈した培地は、ほとんどの場合動作しません。それが低すぎる場合浸透圧を高めるために、D -グルコースを使用してください。 </li></ol></li><li>フェーズは準備時間に起因するシフト スライス標本の時間は非常に重要です。スライスの手順はZT 6月12日12の間に実行された場合、位相でゼロまたは最小限の効果が得られる。すべてのスライスの解離は、準備の間の位相変動に起因する誤差を最小化するために、日周または概日周期の同位相で実行する必要があります。 </li></ol>実行可​​能な変更<ol><li>スライスが開始する前に酸素（95％O 2、5％CO 2）で飽和バッファー、急性SCNの電気生理学の場合、スライスはリンガー（ACSF人工脳脊髄液）で実行する必要があります。さらに、酸素は、電気とシナプス活動は、酸素の電源22に直接依存しているとして、全体のスライス手順の実行中にバッファに連続して追加する必要があります。 </li><li>水平方向と矢状スライスは正常に準備し、SCNを培養することができる。著者は、我々の経験によるとコロナのカットと比較して低い振幅で概日振動を与える水平スライスにおけるルシフェラーゼの録音と経験を、持っている。 </li><li> SCNはrostro - 尾側に長さ約1mmであるとスライシング技術は、ここで説明する中央部にSCNのセクションを取得を対象としています。ただし、複数のSCNのセクションは、SCNの核のより吻側対尾側レベルでの概日遺伝子/タンパク質発現の調査を可能にする、マウスおよびラットの脳から得ることができる。 </li><li>さらに、若い動物、出生後の仔や胚からスライスの培養物は、また、調製することができる。非常に若い子犬の脳と頭蓋骨は非常に柔らかく、敏感であることに注意してください。さらに、視神経は薄いと十分に発達していないです。 SCNの領域が損傷していないように仔の脳を解剖する際はご注意ください。彼らは大きすぎるので、例えば、マイクロ骨鉗子のツールは、仔マウスの頭蓋骨を開くために使用することができます。ファインはさみは若い仔で軟部組織を切断するのに十分です。 </li><li>ここで説明するカバーガラスベースの培養技術は、単一SCNのニューロンでは、CCD / EM - CCDカメラを用いた画像の発光にも使用できます。 </li><li>他の中枢神経系領域とトランスジェニックPER2から末梢器官組織：：LUCの動物が容易にPER2として、分子振動の面で、得られる培養し、分析することができます：：LUCの発現が他の組織の数にも多くの日のために発振し続けると細胞のタイプ10。これらの末梢組織のほとんどは、インスタンスの肝臓のために、生存のための膜を必要としないのではなく、直接媒体11を浴びて培養されています。により、切片にシフトその位相を注意してください、培養と培地交換はSCNと同じ原則に従っていない可能性があります。 </li></ol>意義トランスジェニックマウスとラット系統（：mPER2：LUC、mPer1 - lucを8、10、27）でルシフェラーゼ酵素活性は、タンパク質や遺伝子発現のリズムを反映し、生物発光の記録によって評価することができる。ルシフェラーゼによって生成される生物発光は、微弱な信号を与えるが、バックグラウンド発光は、この方法が有利なこと、ゼロに近いです。また、ルシフェラーゼ分子は不安定で速やかに分解されるためにはpHはありません長期的な興奮照明28の間に表示される可能性難聴、。一緒に、これらのプロパティは、概日研究において非常に有利なルシフェラーゼレポーターの技術を作る長期的な実験を可能にする。

<table><tbody><tr><td>B27 supplement 50x</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Cover glasses</td><td>Menzel-Glaser</td><td>40#1</td><td>&amp;#8776; 40 mm</td></tr><tr><td>Culture membranes</td><td>EMD Millipore</td><td>PICMORG50</td><td>0.4 &amp;#x03BC;m</td></tr><tr><td>DMEM low glucose w/o phenol red</td><td>Sigma-Aldrich</td><td>D2902</td><td>Powder for 1L</td></tr><tr><td>Filter paper</td><td>Whatman, GE Healthcare</td><td>1003 055</td><td>&amp;#8776; 55 mm</td></tr><tr><td>Filtration set</td><td>Corning</td><td>431097</td><td>Pore size 0.22 &amp;#x03BC;mPolyethersulfone</td></tr><tr><td>Forceps </td><td>Allgaier Instrumente</td><td>0203-7-PS</td><td>Dumant #7</td></tr><tr><td>HEPES</td><td>Invitrogen</td><td>15630-056</td><td>100 ml</td></tr><tr><td>HBSS 10x</td><td>Invitrogen</td><td>14065-049</td><td>500 ml</td></tr><tr><td>NaOCH&lt;sub&gt;3&lt;/sub&gt; 7.5%</td><td>Invitrogen</td><td>25080-060</td><td>50 ml</td></tr><tr><td>Luciferin </td><td>Promega Corp.</td><td>E1602</td><td>Beetle luciferin, potassium salt</td></tr><tr><td>Micro rongeur tool</td><td>Allgaier Instrumente</td><td>332-097-140</td><td></td></tr><tr><td>PenStrep 10,000 U/ml</td><td>Invitrogen</td><td>15140-122</td><td>100 ml</td></tr><tr><td>Petri dishes</td><td>Corning</td><td>430165</td><td>&amp;#8776; 35 mm</td></tr><tr><td>PMT</td><td>Hamamatsu Corp.</td><td>H9319-11MOD</td><td></td></tr><tr><td>Disposable scalpels</td><td>Paragon</td><td>P503</td><td>Size 11</td></tr><tr><td>Vacuum filter</td><td>Fisher Scientific</td><td>09-761-5</td><td></td></tr><tr><td>Vacuum grease</td><td>Dow Corning</td><td></td><td>50 g</td></tr><tr><td>Vibratome</td><td>Campden Instruments</td><td></td><td></td></tr></tbody></table>

slice preparation, organotypic tissue culturing and luciferase recording of clock gene activity in the suprachiasmatic nucleus

生理学と行動で毎日のリズムを調整する中心的な概日リズム（〜24時間）クロックは、視床下部前部にある視交叉上核（SCN）に存在する。クロックは、直接網膜と視神経を介して光によって同期化されます。概日振動は、 周期 （ 単位 ）の遺伝子を含む、いわゆる&quot;時計遺伝子&quot;と、そのタンパク質産物の数、負のフィードバックループを相互作用によって生成されます。コアクロックはまた、膜の脱分極、カルシウムやキャンプ1に依存しています。 SCNは、時計遺伝子の発現、代謝活性と自発電気活動の毎日の振動を示しています。驚くべきことに、この内因性の周期的活動は、SCN 2-4の成体組織スライスに保持されます。この方法では、体内時計は簡単にペースメーカー機能の分子、電気生理学的及び代謝の研究を可能にする、in vitroで研究することができます。 SCNは、右の視交叉5の上方に位置する小規模な、よく定義された二国間の構造体です。ラットでは、それぞれの核に〜8.000ニューロンが含まれており、約947ミクロン（長さ、rostrocaudal軸）× 424μmの（幅）× 390μmの（高さ）6の寸法を有する。それは、SCNを識別することができる脳の特定のレベルでの脳のスライスをカットするために必要なSCNを分析する。ここで、我々は、マウスやラットの脳についても同様であるSCN、の解剖とスライスの手順を説明します。さらに、我々はどのように膜7、山崎らによってSCNの組織培養用に開発された技術上organotypically解剖組織培養に示す。8。最後に、我々は、動的なルシフェラーゼレポーターテクノロジー、もともとGeusz ら概測定に使用されたメソッドを使用して時計遺伝子/タンパク質の発現を測定するために使用する方法をトランスジェニック組織を示しています。9。 ：我々はトランスジェニックノックインPERIOD2からSCN組織をここでを使用してください：。ユジンらが生成されるルシフェラーゼマウス10。マウスは、PERIOD（PER）の融合タンパク質を含んで2とホタルの酵素ルシフェラーゼ。ルシフェラーゼは、ルシフェリンの酸化を触媒するときにルシフェラーゼの基質の存在下で翻訳されている場合PER2、すなわちルシフェリン、PER2の発現を生物発光として監視することができます。放出されるフォトンの数は、積極的に生産さPER2蛋白質の量に相関し、そして生物発光リズムは生体 10 で PER2蛋白質のリズムと一致している。この方法ではPER2の発現の周期的な変動が継続的に多くの日中にリアルタイムに監視することができます。我々は組織培養とリアルタイムの生物発光のレコーディングに従うプロトコルは、徹底的に山崎と高橋11で記載されている。

Watch this Scientific Journal Video about Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus at JoVE.com

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

成体マウス視床下部視交叉上核（SCN）、そして文化への迅速な方法器官培養条件下でのSCNの組織を含む準備スライス、の手順が報告されています。さらに、動的なルシフェラーゼレポーターの技術を使用して振動時計遺伝子のタンパク質発現の測定が記述されています。

スライスの準備、器官組織培養と視交叉上核における時計遺伝子の活動のルシフェラーゼ記録

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

slice-preparation-organotypic-tissue-culturing-luciferase-recording

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

35.1K ビュー.  Karolinska Institutet. 成体マウス視床下部視交叉上核（SCN）、そして文化への迅速な方法器官培養条件下でのSCNの組織を含む準備スライス、の手順が報告されています。さらに、動的なルシフェラーゼレポーターの技術を使用して振動時計遺伝子のタンパク質発現の測定が記述されています。

ビデオ: Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

ヒト初代細胞培養物における概日時計遺伝子発現やホルモン分泌のパラレル測定

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

遺伝学

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

新皮質の器官型スライス標本からの全細胞記録

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

神経科学

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

同時電気生理学的記録および視交叉上核ニューロンのカルシウムイメージング

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

マウス神経新生時のノッチシグナル伝達ダイナミクスのリアルタイム生物発光イメージング

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

発生生物学

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

生体内で蛍光レポーターを使用してマウス視交叉上核における時計遺伝子発現のモニタリング

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

単一セルの解像度蛍光は住む幼虫脳文化のショウジョウバエ概日時計のイメージング

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

ルシフェラーゼ生物発光レポーターを用いた遺伝子発現のモニタリング細胞自律的な概日時計のリズム

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

生物学

Long-range Channelrhodopsin-assisted Circuit Mapping of Inferior Colliculus Neurons with Blue and Red-shifted Channelrhodopsins

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, making it difficult to isolate inputs from individual sources with electrical stimulation. However, by using channelrhodopsin assisted circuit mapping (CRACM), this limitation can now be overcome. Here, we report a method to use CRACM to map ascending inputs from lower auditory brainstem nuclei and commissural inputs to an identified class of neurons in the inferior colliculus (IC), the midbrain nucleus of the auditory system. In the IC, local, commissural, ascending, and descending axons are heavily intertwined and therefore indistinguishable with electrical stimulation. By injecting a viral construct to drive expression of a channelrhodopsin in a presynaptic nucleus, followed by patch clamp recording to characterize the presence and physiology of channelrhodopsin-expressing synaptic inputs, projections from a specific source to a specific population of IC neurons can be mapped with cell type-specific accuracy. We show that this approach works with both Chronos, a blue light-activated channelrhodopsin, and ChrimsonR, a red-shifted channelrhodopsin. In contrast to previous reports from the forebrain, we find that ChrimsonR is robustly trafficked down the axons of dorsal cochlear nucleus principal neurons, indicating that ChrimsonR may be a useful tool for CRACM experiments in the brainstem. The protocol presented here includes detailed descriptions of the intracranial virus injection surgery, including stereotaxic coordinates for targeting injections to the dorsal cochlear nucleus and IC of mice, and how to combine whole cell patch clamp recording with channelrhodopsin activation to investigate long-range projections to IC neurons. Although this protocol is tailored to characterizing auditory inputs to the IC, it can be easily adapted to investigate other long-range projections in the auditory brainstem and beyond.

Channelrhodopsin-assisted circuit mapping (CRACM) is a precision technique for functional mapping of long-range neuronal projections between anatomically and/or genetically identified groups of neurons. Here, we describe how to utilize CRACM to map auditory brainstem connections, including the use of a red-shifted opsin, ChrimsonR.

青色および赤色シフトチャネル性肝炎を伴う下コリキュラスニューロンの長距離チャネル性チャネル化補助回路マッピング

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

発光時計レポーターとシロイヌナズナプロトプラストトランスフェにおける概日表現型の迅速分析

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.