作業は、補完代替医療（R21AT00978）のためのNIH /ナショナルセンター（1 - P01 - 002048〜01）（K01 - AT - 002166〜01）、研究資源のためのナショナルセンター（P41RR14075）、精神障害によってサポートされていましたと神経科学ディスカヴァリー研究所（MIND）と脳プロジェクト無償（NS 34189）。

パート1：インフォームドコンセントと被験者の準備健康な成人ボランティアは、手続き、承認審査委員会によると研究のために募集しています。テーマ別インフォームドコンセントは、調査を開始する前に署名されます。 パート2：生理学的モニタリング機器のセットアップ生理学的データが入力チャンネルに記録されたfMRIのトリガ信号を使用してfMRIのスキャンと同期するためにAD楽器PowerLabシステムが可能になります。これは、ECG、呼吸、皮膚コンダクタンスの応答、パルスオキシメトリー、および記録する必要があると、他の生理的信号の入力を持つラップトップベースのシステムです。 <ol><li>呼吸ベルトシステムが使用されている場合、変換器は、走査室とのPowerLabにつながるBNCコネクタに入口を経由してベルト給につながるチューブに、スキャナに隣接する部屋の電源コンセントに接続する必要があります。制御室をスキャン。 </li><li>トリガはfMRIのデータに生理学的データを同期するために使用されている場合は、BNCケーブルは、スキャン結果の制御室でPowerLabにMRIスキャナのトリガ信号を接続する必要があります。 </li><li>監視デバイスを収集します。四ECG電極が必要だけでなく、ECGケーブルは、in vivoのシステムrollcartで互換性のあるMRに接続されます。 </li><li>皮膚コンダクタンスは、スキャン室のブレイクアウトボックスに皮膚コンダクタンスコネクタに差し込みます、測定、およびスキャンの部屋の準備ができて皮膚コンダクタンスゲルを持っているされている場合。 </li></ol>パート3：MRスキャンの準備テーマ<ol><li>基本的な状態は、鍼刺激に対する脳の反応に影響を与えることができるので、スキャンの日に過度の不安や疲労を排除することが重要です。弱さと厄介な反応を避けることができるように、被写体が前の手順に軽い食事を示唆している。 </li><li>スキャナのノイズを抑制するために耳栓を着用マシンで、クッションをサポートによって固定化頭と背臥位になるために主題を指示する。鍼刺激のスキャンの信号/雑音比が一般的なモータや視覚的なタスクに比べて低いため、データはより簡単にヘッドの動きによって破損される傾向にある。サポートは、かかとが体の動きを減らすためにストレッチャーの表面を形成維持するために膝の下に配置されます。ヘッドのコイルは固定され、クッションが快適さのために調整されます。 2時間の平均的なスキャンセッションでは、快適さは途切れないスキャンに非常に重要です。 </li><li>生理的な監視が必要な場合は、件名はスキャナに投入される前に、前のセクションにしたがって調製する必要があります。それは、可能な場合はスキャン中にアイトラッキングと被験者の覚醒状態を監視することが望ましい。 </li><li>被験者の左胸の上部に4 ECG電極を取り付けます（女性被験者に対して、そのようなスキャン制御室にベイにウィンドウにブラインドを下げ、スキャナのドアをシャットダウンするなど、プライバシーの予防措置をとる）。電極にし、生体内のデバイスのケーブルを取り付けます。安全のためにこのケーブルと主題につながるすべての他のケーブルにループを形成せずに、スキャンのテーブルに沿って直接実行する必要があります。生体デバイスの電源をオンにし、ECG信号の品質を確認してください。必要に応じて電極の位置を調整します。 </li><li>導電性ゲルとSCRの電極の井戸を埋める。この特殊なSCRゲルは、皮膚への等張にする必要があります。 Chartソフトウェアで、オープン回路のゼロを実行します。快適であると同時にしっかりと周りにマジックテープをラップすることによって、被験者の手の2つの隣接する遠位指パッドにSCRの電極を取り付けます。 </li></ol>パート4：イメージング鍼刺激に合わせた機能的MR撮像シーケンス。 <ol><li>我々は標準的な二次ヘッドコイルでエコープラナーイメージングのために装備さ1.5または3.0テスラシーメンストリオMRIシステムを（シーメンス、エルランゲン、ドイツ）を使用します。 </li><li>年間の我々の研究は一貫して鍼治療は、脳の複数のレベルで機能的なシステムを広範囲に行動していたことが明らかになった。このような扁桃体、海馬、腹内側前頭前野と下野の分野として脳の底部にある構造は、感受性のアーティファクトの影響を受けやすいです。我々は同じような電界強度を持つとデータ収集のための同様のMRIシーケンスを持つシーメンスのスキャナの二つのモデル間の信号の損失の違いを見つけたとして、これらの地域での信号損失を除外するためにそれぞれのEPI機能的なスキャンを確認することが重要です。 </li><li>薄い脳スライスは、感受性のアーティファクトを減少させるために好ましい。スライス0.6ミリメートルまたは矢状または軸方向、0.75 mmのギャップを持つ厚さ3mm、イメージングシステムのメモリ容量に応じて、脳幹や小脳を含めた脳全体をカバーするために使用されます。他の方向とスライス厚が部分的な脳のイメージングに使用することができます。我々はこれらの問題に支払う特別な注意はより多くのに貢献する脳の基部にある構造物に対する鍼の効果の一貫性のある結果。 </li><li>機能的なスキャンは、T2 *-加重グラディエントエコーシーケンス（TR / TE 30分の4000ミリ秒、FOV 200ミリメートル、マトリックス64 × 64、フリップ角90 °、スライス厚3.0ミリメートル、ギャップ0.6 mm形）で取得されています。我々は10分で150時間点の合計を取得しました。画像のコレクションは、fMRIの信号の平衡を可能にするには、4ダミースキャンが付いています。短いTEは、感受性のアーチファクトを低減します。比較的長いTRは、高い空間分解能で脳全体のカバレッジを可能にします。 </li></ol>パート5：最初のローカライザおよび解剖学的スキャン<ol><li>鍼治療と機能的なスキャンが実行される前に、ローカライザースキャン、高解像度の解剖学のスキャン、およびT1強調3D MP - RAGEは、正確な位置決めと解剖学的健全性を保証するために取得されます。この時点で一般的な観察は、不要なスキャンが実行されていないことを保証することができます。ローカライザー、非常に短いスキャンは、脳全体がスキャンに含まれていることを保証し、AC - PCラインでそれを整列することができます。疑わしい異常がある場合は、研究者が訓練を受けた放射線科医に画像を送信しますが、対象へのスキャンでは何かについての主張を行うべきではありません。 </li><li>最初のスキャンは約20分の合計を取る。それはスキャンを継続するために有用となる場合について簡単に閲覧、調査を指示することができます。これは、サブジェクトと話すと、彼らはまだ満足していると磁石にいるからどんな副作用が発生していないことを確認する良い機会です。 </li></ol>パート6：fMRIによるモニタリングと鍼治療治療（マニュアル針操作と電気刺激）の2つの形式が、このビデオは、臨床使用のはるかに長い歴史を持っているマニュアル鍼、これに限定されます。鍼刺激は、滅菌、1回使用のみ鍼を用いて、脚に手で古典的なツボLI4、徒歩でLV3またはST36に重複して実行で投与される。 <ol><li> 、リラックス彼らの目を閉じておくと、スキャン中に運動を控えるよう被験者に指示します。有害な刺激を避けるために、我々は、特に鍼の感覚のどれかが8のスコアに近づく場合は、1つの指を高めるために被験者に指示し、任意の次数の鋭い痛みの場合には二本の指を上げる。 </li><li>針を挿入した後、挿入とデータ収集のための操作の力の深さを選択できるように、針操作対象の感度と耐性をプレテスト。 </li><li> 10分機能的イメージングの間に、針が）M1とM2の間に2分間回転させ、そして残りの期間R1（2分）の間の場所で左、R2（3分）、R3（1分）。鍼灸師でもその動きが結果に影響を及ぼす可能性があるため、刺激期間の前または後に接近または直接主題を残さないことを確認します。 （パラダイム、図1） <img src="/files/ftp_upload/1190/1190fig1.n.jpg" alt="図1" /> 図1。鍼fMRIのパラダイム。 </li><li>鍼刺激が完了すると、鍼治療の針は操作に抵抗する力を記録します。各スキャンの終了時に、別のスタッフの研究者は、1〜10のスケールで経験したグレード感覚に従うように要求します。リスト、痛み、苦痛、圧力、膨満感、重苦しさ、しびれ、うずき、鈍い痛み、鋭い痛みが、鍼治療のコミュニティという文学の痛みのアンケートより前の実験データの経験に基づいている。心理物理学的応答は、これらのレコードに基づいて、 鋭い痛みと混合徳啓の典型的な徳奇のグループと別のグループに分類される。 </li><li>彼らが快適に感じると、彼らが起きていることを確認するためのスキャンの間に被験者に話す。自分の体のどの部分が痙攣し始めたり、不快に感じる場合は、サイト上の研究スタッフは、不快感を和らげるために適切な措置をとるべきである。被験者によって生成された動きは、データの破損の原因となります。時折被験者は血液動態反応に影響を及ぼすような、スキャン中に眠りに落ちることがあります。 </li></ol>パート7：データ処理<ol><li>解析を複数のレベルで実行することができます。一般的な線形モデルでは、タスク中の脳のすべてのボクセルで％BOLD MR信号の変化を決定するためにベースラインとして、残りの期間を使用しています。また、これらの変化の統計的有意性を決定し、特定のp -値で3連続したボクセルに到達しないBOLDデータを削除します。 </li><li> one神経学の領域と脳の残りの部分との間の機能的な接続を表示するためにシードベースの相互相関分析を実行します。これは、参照領域を選択し、脳内の他のすべてのボクセルのそれとの時間経過を比較することによって行われます。このような独立成分分析、ファジィクラスター分析、構造方程式モデリング、グレンジャーの因果性のような他の分析を適用することができます。 </li><li>生理学的データができます鍼の刺激に関連したウィンドウ内に応答を平均することにより分析する。例えば、心電図のデータは、心拍数と心拍変動の分析に続いてQRS波のタイミングのための注釈を付けることができます。 </li></ol>パート8：結果<img src="/files/ftp_upload/1190/1190fig2.n.jpg" alt="図2" /> 図2。鍼徳啓対右LI4、ST36とLV3（52 runs/37科目）における触覚刺激で血行動態の応答。 MPFC（内側前頭前野）における不活性化構造のクラスター（1）、MPC（内側頭頂皮質）（2）、MTL（内側側頭葉）（3）、触覚刺激に比べて（図）鍼でより顕著であった（図B）。鍼で右BA22/21の広範な非活性化（4）触覚刺激では観察されなかった。感覚BA43（5）と連合野（6）鍼（図示せず）に比べて触覚刺激でより多くの活性化を示した。興味深いことに、右前島、（7）鍼ではなく、触覚刺激で活性化した。 （P &lt;0.0001）。 <img src="/files/ftp_upload/1190/1190fig3.n.jpg" alt="図3" /> 図3。鋭い右LI4、ST36での鍼刺激時の痛み（52 runs/29科目）とLV3、MPFCの著名な非活性化（1）、MPC（と混合徳琪 （52 runs/37科目）対徳啓における血行動態反応2）とMTL（3）徳啓ない痛み（図A）に見られる痛みの存在（図B）に減弱した。痛みで、感覚運動と関連皮質の活性化（4）より顕著になり、そしてそのようなMIDC / SMA（5）、postC_BA23（6）、エイミー（7）、および小脳虫部のような大脳辺縁系とparalimbic地域のサブセット（ 8）活性化となった。前部と後部島（10）の小さい領域は痛みで二国間で活性化されたまま右前島（9）著しく徳啓で活性化した。 P &lt;0.0001。 <img src="/files/ftp_upload/1190/1190fig4.n.jpg" alt="図4" /> 図4デフォルトモードのネットワーク（DMN）と鍼治療における大脳辺縁系- paralimbic -新皮質ネットワーク（LPNN）のオーバーラップ、。GLM分析（図A）でとモデルフリーファジィクラスター分析（図BによるLPNNのBOLD応答）LI4における鍼刺激の間に、ST36とLV3はDMN（Fig.C、シュルマンら、1997から引用）とほぼ同じパターンを示す。コヒーレント信号の減少と地域のクラスターは、内側前頭前野（1）、postero -内側皮質（2）と側頭葉（3）に表示されます。 P &lt;0.0001。

<ol>
	<li>Hui, K. K. S., Liu, J., Marina, O., Napadow, V., Haselgrove, C., Kwong, K. K., Kennedy, D. N., Makris, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+integrated+response+of+the+human+cerebro-cerebellar+and+limbic+systems+to+acupuncture+stimulation+at+ST+36+as+evidenced+by+fMRI.">The integrated response of the human cerebro-cerebellar and limbic systems to acupuncture stimulation at ST 36 as evidenced by fMRI.</a> Neuroimage. 27 (3), 479-496 (2005).</li><li>Hui, K. K. S., Marina, O., Claunch, J. D., Nixon, E. E., Fang, J., Liu, J., Li, M., Napadow, V., Vangel, M., Makris, N., Chan, S. T., Kwong, K. K., Rosen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acupuncture+mobilizes+the+brain's+default+mode+and+its+anti-correlated+network+in+healthy+subjects.">Acupuncture mobilizes the brain's default mode and its anti-correlated network in healthy subjects.</a> Brain Res. 1287, 84-103 (2009).</li><li>Napadow, V., Dhond, R. P., Purdon, P., Kettner, N., Makris, N., Kwong, K. K., Hui, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+acupuncture+fMRI+in+the+human+brainstem+with+heart+rate+variability.">Correlating acupuncture fMRI in the human brainstem with heart rate variability.</a> Conf Proc IEEE Eng Med Biol Soc. 5, 4496-4499 (2005).</li><li>Shulman, G. L., Corbetta, M., Buckner, R. L., Raichle, M. E., Fiez, J. A., Miezin, F. M., Petersen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-down+modulation+of+early+sensory+cortex.">Top-down modulation of early sensory cortex.</a> Cereb. Cortex. 7 (3), 193-206 (1997).</li></ol>

不注意な有害刺激の交絡影響から分離を可能にするために適切なテクニック、鍼治療の経験と大規模なサンプルサイズの雇用は、鍼fMRIのデータの収集と解釈において重要である。いくつかの古典的なツボの我々の研究は、鍼治療は辺縁系ネットワーク、人間の脳の重要な本質的な規制システムを調節することを示している。ネットワークは、安静時の脳のデフォルトモードと大幅に重複する...

monitoring acupuncture effects on human brain by fmri

機能的MRIは、BOLD応答と人間の脳の機能的結合に対する鍼治療の効果を研究するために使用されます。結果は、鍼治療は、辺縁系 - paralimbic -新皮質のネットワークを動員し、脳の複数のレベルで、そのその反相関感覚/ paralimbicネットワークが血液動態反応は心理物理学的応答に影響されることを示している。生理学的モニタリングは、自律神経機能の周辺機器の応答を調べるために実施することができる。このビデオはLI4（河谷）で行われた研究は、ST36（zusanli）とLV3（太衝）、一般的に調節し、痛みの軽減の行動のために使用されている古典的なツボを説明します。鍼治療の研究にfMRIのアプリケーションで注意が必要ないくつかの問題が指摘されています。

Watch this Scientific Journal Video about Monitoring Acupuncture Effects on Human Brain by fMRI at JoVE.com

Monitoring Acupuncture Effects on Human Brain by fMRI

FMRIと生理学的モニタリングは、中枢および末梢神経系に対する鍼の効果を研究するために使用されます。鍼治療はおそらく末梢神経系における自律神経作用に関連し、神経学的活性を調節するために、デフォルトモードのネットワークを持つ偉大なオーバーラップで、辺縁系 - paralimbic -新皮質のネットワークを動員。

fMRIによるヒト脳機能に対する鍼の効果のモニタリング

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

monitoring-acupuncture-effects-on-human-brain-by-fmri

Research

JoVE Journal

Biology

15.2K ビュー.  Massachusetts General Hospital and Harvard Medical School. FMRIと生理学的モニタリングは、中枢および末梢神経系に対する鍼の効果を研究するために使用されます。鍼治療はおそらく末梢神経系における自律神経作用に関連し、神経学的活性を調節するために、デフォルトモードのネットワークを持つ偉大なオーバーラップで、辺縁系 - paralimbic -新皮質のネットワークを動員。

ビデオ: Monitoring Acupuncture Effects on Human Brain by fMRI

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人間のDNA修復タンパク質の遺伝的研究は、モデルシステムとして酵母を使用

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

生物学

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

偏上皮細胞へのプラスミドのマイクロインジェクションによる低分子量GTPaseの機能を解析する

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

機能イメージングによるヒト聴覚皮質上のシータバースト刺激の後遺症のマッピング

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

の検討<em&gt;ショウジョウバエ</em光学顕微鏡で&gt;幼虫気管ターミナルセル

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

酸化ストレスに赤/近赤外光療法の波長および強度の範囲の効果の評価のための手法<em&gt;インビトロ</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

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 ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮細胞接着性の定量<em&gt;インビトロ</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

使用して小胞体カルシウムの恒常性の監視<em&gt;ガウ</em&gt;ルシフェラーゼSERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

enChIPによって特定のゲノム領域と関連する分子の同定の単離

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...