PV 5のリアルタイムfMRIをセットアップするためのAFNIスクリプトを共有してくれたD.Chen博士とC.Yen博士、およびソフトウェアサポートを提供してくれたAFNIチームに感謝します。この研究は、NIHブレインイニシアチブの資金提供(RF1NS113278-01、R01 MH111438-01)、およびドイツ研究財団(DFG)のマルティノスセンターへのS10機器助成金(S10 RR023009-01)、およびマックスプランク協会からの内部資金提供によってサポートされました。

この研究は、ドイツ動物福祉法(TierSchG)および動物福祉実験動物条例(TierSchVersV)に従って実施されました。ここに記載されている実験プロトコルは、倫理委員会(§15 TierSchG)によってレビューされ、州当局(ドイツ、バーデンヴュルテンベルク州テュービンゲンのRegierungspräsidium)によって承認されました。
1. 小動物研究のためのBOLD-fMRI実験セットアップの準備
<ol><li>コンソールソフトウェアの電源を入れて、イメージングパラメータを制御し、MRIデータを取得します。 注:提案されたリアルタイムfMRIセットアップは、AFNIの画像処理機能と並行して、コンソールソフトウェア(バージョン6)のマクロ機能を利用して実装されています。</li>
	<li>ワークスペースエクスプローラーでMRシーケンス(位置、ローカライザー、リラクゼーションエンハンスメント(RARE)による高速取得、3Dエコープレーナイメージング(EPI)など)を検索し、スキャンリストにドラッグアンド追加します。 注:位置とローカライザーのシーケンスは、脳内の関心領域(ROI)を特定するために使用されます。まれなシーケンスは、解剖学的スキャンに使用されます。3D EPIシーケンスは、動的なBOLD応答を測定するために使用されます。</li>
	<li>定義済みのマクロスクリプト "Setup_rt3DEPI" と "Feed2AFNI_rt3DEPI" をマクロスクリプトパスに配置します (例: "/opt/(PV バージョン)/prog/curdir/(ユーザー名)/ParaVision/macros")。「データ再構築」ユーザーインターフェイスメニューの3D EPI再構築オプション「プレイメージシリーズアクティビティ」と「マクロの実行」を有効にし、事前定義されたマクロスクリプト「Setup_rt3DEPI」をリンクしてから、「スキャン」ボタンをクリックします。 注: マクロ スクリプトは補足 ファイルに含まれています。</li>
	<li>リアルタイムのBOLD-fMRI分析と視覚化のためのAFNIソフトウェアをインストールします。</li>
</ol>2.カテーテル法と換気手術
<ol><li>図1に示すように、人工呼吸器と体温計、血圧、呼吸記録などの生理学的状態監視システムをセットアップします。人工呼吸器で60±1呼吸/分の一定周波数を設定し、フィードバック制御セット付きのMR対応加熱パッドを使用して37°Cの温度を設定します。</li>
	<li>誘導のために5%イソフルランを含むチャンバーで成体の雄Sprague-Dawleyラット(300-600g)を麻酔し、気化器から手術用の2-2.5%イソフルランを送達する。後足をつまんで麻酔の深さを確認し、離脱反応がないことを確認します。</li>
	<li>換気のために14 Gのプラスチックカニューレで動物を挿管します(70%の空気と30%の酸素の混合物で60 ± 1呼吸/分)。潮汐終末二酸化炭素(CO2)を25±5 mmHg29の範囲になるように調整します。 注:挿管は、fMRI実験を通じて適切なCO2 レベルを維持するために重要です。</li>
	<li>動物を手術台の上で仰臥位にし、電気かみそりで太ももを剃ります。そして、手術用ハサミで剃った皮膚を切開します。 注:切開の長さは縦方向に約1〜2 cmです。</li>
	<li>カテーテル挿入のために切開領域の下に大腿動脈と静脈を見つけ、個々の大腿動脈と静脈を周囲の組織から分離します。</li>
	<li>分離した大腿動脈の片側を外科用縫合糸で固定し、反対側をマイクロブルドッグ鉗子で保持します。次に、大腿動脈の結ばれた領域の間に小さな切開を行います。</li>
	<li>小さな切開部から大腿動脈にカテーテルを挿入し、カテーテルと動脈を外科用縫合糸で結びます。生理学的モニタリングシステムで動脈血圧を常に監視し、80〜120 mmHgの範囲になり、動脈血ガスを定期的に測定して、スキャン中に最小90 mmHgのpO 2と30〜45 mmHgのpCO2を維持します。 注:このカテーテル法は、fMRI実験中に動脈血圧を監視するために重要です。</li>
	<li>大腿静脈の両端を絹編組外科用縫合糸で固定します。次に、大腿静脈の結ばれた領域の間に小さな切開を行います。鉗子を使用して縫合を行います。 注:縫合糸のサイズは約1〜2 cmです。</li>
	<li>カテーテルを大腿静脈に挿入します。カテーテルと静脈を外科用縫合糸で結びます。 注:このカテーテル法は、静脈を通してアルファクロラロースを投与し、fMRI実験中に麻酔レベルを調整するために重要です。動物が十分に麻酔されていない場合、それは自発的に呼吸し始めます。この場合、呼吸運動アーチファクトを避けるために、より多くのα-クロラロースを投与する必要があります。</li>
	<li>剃った皮膚の外科的切開部を縫合します。外科的処置が完了したら、大腿静脈に接続されたカテーテルに~80 mg / kgの投与量のアルファクロラロースのボーラスを注入して動物を麻酔し続け、同時にイソフルラン投与を中止します。</li>
</ol>3.動物をMRIスキャナーの中に置きます
<ol><li>2.10ステップが完了したらすぐに麻酔をかけた動物をMRIスキャナーに移し、カスタムメイドのクレードルに固定します。</li>
	<li>動物の体温を監視するために、動物にリアルタイムのフィードバック直腸体温計を挿入します。温度を制御するために動物の胴体の下に加熱パッドを置きます。MRIスキャン中は体温を37.0±0.5°Cに維持してください。</li>
	<li>筋弛緩剤であるパンクロニウム(~2 mg/kg/h)の混合物に~25 mg/kg/h溶液を含むα-クロラロースを継続的に送達し、動物の麻酔を維持し、fMRI画像のモーションアーチファクトを低減します。生理状態に応じて薬剤の量と換気速度を調整して血圧と呼吸を監視します。</li>
	<li>fMRI実験中の乾燥を防ぐために、動物の目に眼科用軟膏を投与します。頭の動きのアーチファクトを避けるために、2つのイヤーバーで動物の頭を安全に固定します。</li>
	<li>トランシーバーの表面コイルをヘッドに固定します。MRI測定の前に、コイルをヘッドのラーム周波数(たとえば、599 Tで14.1 MHz)に調整して一致させます。 注:ここでは、直径22mmのコイルを使用してラットの脳全体をカバーしています。</li>
	<li>指1と4の間の前足の皮膚に一対の針電極を挿入し、サージカルテープで固定します。そして、これらの電極30に刺激入力ケーブルを接続した後、刺激が正しく動作することを確認する。</li>
	<li>動物をMRIボアに挿入し、ほぼアイソセンターに置きます。</li>
</ol>4. 解剖学的MR画像の測定
<ol><li>メインユーザーインターフェイスのキャリブレーションメニューボタンをクリックします。 調整プラットフォームの ユーザーインターフェイスで次の項目をクリックしてMRIシステムのキャリブレーションを実行します(コンソールソフトウェアの [ヘルプ ]メニューを参照)。 基本的な共振周波数を見つけ、RFパルスパワーを校正し、最適なレシーバゲインを設定し、シミングのために動物のB0マップを測定し、 非局在自由誘導減衰(FID)積分に基づいてグローバルリニアシムを実行します。 注: この手順にかかる時間は 2 分未満です。</li>
	<li>「スキャン」ボタンをクリックして位置シーケンスを実行し、MRIボア内の動物の頭の位置を見つけます。ヘッドがアイソセンターにない場合は、ヘッドがアイソセンターに配置されるまでクレードルを前後に動かしながらヘッドの位置を調整します。</li>
	<li>「スキャン」ボタンをクリックしてローカライザーシーケンスを実行し、ヘッド内のROIを特定します。マップシムを選択し、ローカライザー画像で脳全体をカバーするようにシムボリュームのROIを定義してから、「シムアップ」オプションを使用して高次(たとえば、2次または3次)シムを実行し、ROIでの主磁場(B0)の不均一性を低減します。 注:高次シミングは、EPIシーケンスを使用する場合のBOLD-fMRIデータの品質を向上させるための重要なステップです。</li>
	<li>「スキャン」ボタンをクリックしてT2強調RAREシーケンスを実行し、冠状視野で脳全体をカバーする解剖学的画像を取得します(たとえば、次のシーケンスパラメータが使用されます:繰り返し時間(TR)4000ミリ秒、実効エコー時間(TE)36.1ミリ秒、マトリックス128 x 128、視野(FOV)19.2x19.2 mm2、スライス数32、スライス厚さ0.3 mm、 まれ因子8)。 注:次のリアルタイムfMRI視覚化ステップでは、解剖学的画像を使用して3D EPI画像をテンプレートとして登録します。</li>
</ol>5. リアルタイムfMRIソフトウェアのセットアップとfMRI応答の可視化
<ol><li>ターミナルウィンドウを開き、次のコマンドを使用してリアルタイムAFNIプラグインパスに移動します。 cd /home/(ユーザ名)/rt_afni 注: AFNI プラグインスクリプト "afni_rt" は 補足ファイルに含まれています。</li>
	<li>以下のコマンドとオプションを使用して、リアルタイムプラグインでAFNIソフトウェアを実行します。 アフニ -RT -yestplugouts -DAFNI_REALTIME_MP_HOST_PORT=ローカルホスト:(ポート番号) -DAFNI_REALTIME_Graph=リアルタイム -DAFNI_FIM_IDEAL=(パラダイム) 注:前者の場合、コードは外部プログラムがAFNIとデータを交換することを許可し、後者の場合、リアルタイムプラグインはユーザー定義のローカルホストとポートへのTCPソケットを開こうとします。3番目と4番目のケースでは、コードはfMRIデータの時間経過をリアルタイムでプロットし、リアルタイムfMRIデータが取得されたときにそれぞれfMRI時間経過にユーザー定義パラダイムの時間経過をプロットします。詳しくは https://afni.nimh.nih.gov/pub/dist/doc/program_help/README.environment.html をご確認ください。</li>
	<li>以下のオプションを使用して、 図 2 に示すように、コマンド "Dimon" を使用して定義された今後の AFNI BRIK ファイルをモニターします。 Dimon -tr (TR of EPI) -nt (NRerepeats of EPI) -rt -quit -infile_patternリアルタイム*。ブリク -file_type AFNI 注:「Dimon」は、リアルタイムプラグインを実行する「-rt」と「-infile_pattern(データ名)のオプションを使用して、AFNI画像ファイルのリアルタイム取得を監視するコマンドです。BRIK -file_type AFNI」を使用すると、プラグインは特定のBURKファイルを読み取り、表示とフォーマットのためにAFNIに送信できます。詳細については、https://afni.nimh.nih.gov/pub/dist/doc/program_help/Dimon.html を確認してください。</li>
	<li>「pvcmd」コマンドを次のオプションで使用します。 pvcmd -a JMacroManager JMMExecuteMacro -category $USER -macro Feed2AFNI_rt3DEPI 注:このコードは、EPI取得のために「スキャン」ボタンをクリックした直後にバックグラウンドマクロスクリプト「Feed2AFNI_rt3DEPI」を実行するためのマクロスクリプト「Setup_rt3DEPI」に存在します。</li>
	<li>「exec pvcmd」コマンドを以下のオプションとともに使用して、EPI取得パラメータを取得します。 exec pvcmd -a ParxServer -r ParamGetValue -psid $ParSpaceId -param (PVM parameters of EPI) -id 10 -args $AcqKey $ParSpaceId $ProcnoPath</li>
	<li>「exec to3d」コマンドを以下のオプションとともに使用すると、バックグラウンドマクロスクリプト「Feed2AFNI_rt3DEPI」でEPI生データをリアルタイムでAFNIファイルに変換します。 exec to3d -omri -xFOV $FOV_X -yFOV $FOV_Y -zFOV $FOV_Z -prefix $LastVolName $ImgFormat$Path2dseq</li>
	<li>EPIの幾何学的情報が解剖学的方向と一致していることを確認してください。 注: 図2に示すように、各3DボリュームデータがTRごとに保存されるたびに、視野(FOV)やマトリックスサイズなどの幾何学的情報を使用して「to3d」AFNIコマンドが自動的に実行され、fMRI生データを1つのAFNI BRIKデータに変換します。画像の向きは、「to3d」の幾何学的情報パラメータで変更できます。詳しくは https://afni.nimh.nih.gov/pub/dist/doc/program_help/to3d.html をご確認ください。</li>
	<li>電気刺激アイソレーターをオンにし、刺激ブロックを使用して1回の誘発fMRI研究(例:3Hz、4秒パルス幅300us、2.5mA)の電気前足刺激を実行します。 注:ここで、ブロック設計パラダイムは、10回の刺激前スキャン、3回の刺激スキャン、および12回の刺激間スキャン(エポックあたり15回のスキャン)で構成されています。</li>
	<li>BOLD-fMRIスタディの「スキャン」ボタンをクリックして、T2*強調3D EPIシーケンスを実行します(たとえば、TR/TE 1500/14ミリ秒、マトリックス64 x 64 x 32、FOV 19.2 x 19.2 x 9.6 mm 3、分解能300 x 300 x 300 μm3)。 注:「スキャン」ボタンをクリックするとすぐに、事前定義されたマクロスクリプトを使用して生データの監視と処理がリアルタイムで行われます。1つのAFNI BRIKデータセットが変換されると、3D EPI画像のボクセル単位のタイムコースグラフがAFNIソフトウェアに表示され、TRごとに自動的に更新されます。</li>
	<li>解剖学的レア画像の上にEPI画像をオーバーレイするには、手順5.6のようにコマンド「to3d」を使用してレア画像をAFNI BRIKデータセットに変換し、次のオプションを指定して「align_epi_anat.py」AFNIスクリプトを使用してEPI画像を解剖学的画像に登録します。 align_epi_anat.py -アナト anatomy_template_al+オリジナル -エピ エピ.$(エピデータ番号)+オリジナル -epi_base 1 -サフィックス _volreg -rat_align -コスト LPA -エピ2アナト 注:詳細については、https://afni.nimh.nih.gov/pub/dist/doc/program_help/ align_epi_anat.py.htmlを確認してください。</li>
	<li>BOLD応答の関数マップを処理するには、次のオプションを指定して「3dDeconvolve」コマンドを使用して、特定の刺激時系列を持つ3D+時間データセットのデコンボリューションを計算します。 3dデコンボルブ -input (入力ファイル名)+オリジナル -nfirst 0 -polort 3 -num_stimts 1 -stim_times 1 (刺激パラダイムファイル名) 'BLOCK(4,1)' -stim_label 1 フォアポー -tout -fout -rout 注:空間スムージングや時間フィルタリングなどの画像処理ステップは、カスタマイズされたAFNIデータ処理スクリプトに組み込まれています。詳しくは https://afni.nimh.nih.gov/afni/doc/help/3dDeconvolve.html をご確認ください。</li>
	<li>BOLD信号の機能マップを視覚化するには、AFNIソフトウェアでインタラクティブなクラスタリングを使用します。「オーバーレイの定義」オプションを開き、AFNIユーザーインターフェイスメニューから「クラスター」機能を使用します。</li>
	<li>最後のfMRIスキャンの後、動物をMRIスキャナーから取り出し、承認されたプロトコルに従って安楽死させます。 注:リアルタイムfMRIデータの処理には、AFNIの画像処理機能と最新のコンソールソフトウェアのマクロ機能を使用しました。マクロ関数の詳細情報と説明は、コンソールソフトウェアのヘルプメニューから見つけることができます。AFNIソフトウェアはフリーウェアであり、NIMH-AFNIのウェブサイトから直接ダウンロードできます。AFNIとコンソールシステム間のリンクを構築するための関連スクリプトが添付されています。</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+functional+magnetic+resonance+imaging.">Real-time functional magnetic resonance imaging.</a> Methods. 25 (2), 201-220 (2001).</li><li>Posse, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+measure+single-event+related+brain+activity+using+real-time+fMRI:+Feasibility+of+sensory,+motor,+and+higher+cognitive+tasks.">A new approach to measure single-event related brain activity using real-time fMRI: Feasibility of sensory, motor, and higher cognitive tasks.</a> Human Brain Mapping. 12 (1), 25-41 (2001).</li><li>Decharms, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reading+and+controlling+human+brain+activation+using+real-time+functional+magnetic+resonance+imaging.">Reading and controlling human brain activation using real-time functional magnetic resonance imaging.</a> Trends in Cognitive Sciences. 11 (11), 473-481 (2007).</li><li>Bruhl, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+Sense+of+Real-Time+Functional+Magnetic+Resonance+Imaging+(rtfMRI)+and+rtfMRI+Neurofeedback.">Making Sense of Real-Time Functional Magnetic Resonance Imaging (rtfMRI) and rtfMRI Neurofeedback.</a> International Journal of Neuropsychopharmacology. 18 (6), (2015).</li><li>Cox, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFNI:+Software+for+analysis+and+visualization+of+functional+magnetic+resonance+neuroimages.">AFNI: Software for analysis and visualization of functional magnetic resonance neuroimages.</a> Computers and Biomedical Research. 29 (3), 162-173 (1996).</li><li>Liou, W. W., Goshgarian, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+the+effect+of+chronic+phrenicotomy+on+the+induction+of+the+crossed+phrenic+phenomenon.">Quantitative assessment of the effect of chronic phrenicotomy on the induction of the crossed phrenic phenomenon.</a> Experimental Neurology. 127 (1), 145-153 (1994).</li><li>Shih, Y. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+differentially+modulates+neuronal+and+vascular+contributions+to+the+BOLD+signal.">Anesthesia differentially modulates neuronal and vascular contributions to the BOLD signal.</a> Neuroimage. 195, 89-103 (2019).</li><li>Wu, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+isoflurane+anesthesia+on+resting-state+fMRI+signals+and+functional+connectivity+within+primary+somatosensory+cortex+of+monkeys.">Effects of isoflurane anesthesia on resting-state fMRI signals and functional connectivity within primary somatosensory cortex of monkeys.</a> Brain and Behavior. 6 (12), 00591 (2016).</li><li>Liu, X., Zhu, X. 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Sascha KöhlerはBruker BioSpin MRI GmbHの従業員です。

fMRI信号のリアルタイムモニタリングは、実験者が動物の生理機能を調整して機能マッピングを最適化するのに役立ちます。覚醒している動物における運動アーチファクト、ならびに麻酔効果は、fMRI信号の変動性を媒介する主要な要因であり、信号の生物学的解釈をそれ自体で混乱させる31,32,33,34,35,36,37,38<sup class="...

機能的磁気共鳴画像法(fMRI)は、血行動態応答1、2、3、4、5、6、7、8、9、例えば、血中酸素レベル依存性(BOLD)、脳内の神経活動に関連する脳血液量および流量信号を測定する非侵襲的方法である。動物実験では、血行動態信号は、麻酔10、覚醒している動物のストレスレベル11、ならびに潜在的な非生理学的アーティファクト、例えば、心拍動および呼吸運動12、13、14、15によって影響を受ける可能性がある。タスク関連および安静状態機能ダイナミクスおよび接続性マッピング16、17、18、19のためのfMRI信号の遡及的分析を提供するために多くの後処理方法が開発されているが、動物の脳20におけるリアルタイムの脳機能マッピングソリューションおよび瞬間的な読み出しを提供する技術はほとんどない(そのほとんどは、主にヒト脳マッピング21に使用される、 22,23,24,25,26,27)。特に、この種のリアルタイムfMRIマッピング法は、動物実験では欠けています。fMRIプラットフォームを構築して、リアルタイムの脳状態依存的な生理学的病期の調査を可能にし、動物の脳機能研究にリアルタイムのバイオフィードバック刺激パラダイムを提供する必要があります。
本研究では、MRIコンソールソフトウェアのカスタマイズされたマクロ機能を備えたリアルタイムfMRI実験セットアップを示し、麻酔をかけたラットの一次前足体性感覚皮質(FP-S1)における誘発されたBOLD-fMRI応答のリアルタイムモニタリングを示します。このリアルタイムセットアップにより、既存の神経画像解析ソフトウェアである機能神経画像解析(AFNI)28を使用して、機能マップで進行中の脳の活性化と個々の時間コースをボクセル単位で視覚化できます。動物実験のためのリアルタイムfMRI実験セットアップの準備は、プロトコルに記載されています。動物のセットアップに加えて、画像処理スクリプトと並行して、最新のコンソールソフトウェアを使用してリアルタイムfMRI信号の視覚化と分析を設定するための詳細な手順を提供します。要約すると、動物実験用に提案されたリアルタイムfMRIセットアップは、MRIコンソールシステムを使用して動物の脳内の動的fMRI信号を監視するための強力なツールです。

<table>
	<tbody>
		<tr>
			<td>14.1T Bruker MRI system</td>
			<td>Bruker BioSpin MRI GmbH</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>A365 Stimulus Isolator</td>
			<td>World Precision Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>AcqKnowledge Software</td>
			<td>Biopac</td>
			<td>RRID:SCR_014279, http://www.biopac.com/product/acqknowledge-software/</td>
			<td></td>
		</tr>
		<tr>
			<td>AFNI</td>
			<td>Cox, 1996</td>
			<td>RRID:SCR_005927, http://afni.nimh.nih.gov</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2SMO (ETCO2/SpO2 Monitor), Model 7100</td>
			<td>Novametrix Medical Systems Inc</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>CP-Pharma</td>
			<td>Cat# 1214</td>
			<td></td>
		</tr>
		<tr>
			<td>Master-9</td>
			<td>A.M.P.I</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoliter Injector</td>
			<td>World Precision Instruments</td>
			<td>Cat# NANOFIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Pancuronium Bromide</td>
			<td>Inresa Arzneimittel</td>
			<td>Cat# 34409.00.00</td>
			<td></td>
		</tr>
		<tr>
			<td>ParaVision 6</td>
			<td>Bruker BioSpin MRI GmbH</td>
			<td>RRID:SCR_001964</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>Cat# 10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat: Sprague Dawley rat</td>
			<td>Charles River Laboratories</td>
			<td>Crl:CD(SD)</td>
			<td></td>
		</tr>
		<tr>
			<td>SAR-830/AP Ventilator</td>
			<td>CWE</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-chloralose</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# C0128-25G;RRID</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time fmri brain mapping in animals

動的fMRI応答は、麻酔下または覚醒状態の動物の生理学的条件によって大きく異なります。私たちは、取得中にfMRI応答を瞬時にモニターするように実験者を導くリアルタイムfMRIプラットフォームを開発し、動物の生理機能を変更して動物の脳で目的の血行動態応答を達成することができます。リアルタイムfMRIセットアップは、14.1Tの前臨床MRIシステムに基づいており、麻酔をかけたラットの一次前足体性感覚皮質(FP-S1)における動的fMRI応答のリアルタイムマッピングを可能にします。リアルタイムfMRIプラットフォームは、fMRI信号の変動につながる交絡源を調査するための遡及的分析の代わりに、カスタマイズされたマクロ機能とMRIシステムの一般的な神経画像解析ソフトウェアを使用して動的fMRI応答を識別するためのより効果的なスキームを提供します。また、動物の脳機能研究のための即時のトラブルシューティングの実現可能性とリアルタイムのバイオフィードバック刺激パラダイムを提供します。

図3と図4は、代表的なリアルタイムボクセル単位のBOLD-fMRIタイムコースと、電気前足刺激(3 Hz、4 s、パルス幅300 us、2.5 mA)を使用した関数マップを示しています。fMRI設計パラダイムは、10回の刺激前スキャン、3回の刺激スキャン、および合計8回のエポック(130回のスキャン)の12回の刺激間スキャンで構成されています。合計スキャン時間は3分15秒(195?...

Watch this Scientific Journal Video about Real-Time fMRI Brain Mapping in Animals at JoVE.com

Real-Time fMRI Brain Mapping in Animals

動物の脳機能マッピングは、リアルタイムの機能的磁気共鳴画像法(fMRI)実験セットアップの恩恵を受けることができます。動物MRIシステムに実装された最新のソフトウェアを使用して、小動物fMRIのリアルタイムモニタリングプラットフォームを確立しました。

動物におけるリアルタイムfMRI脳マッピング

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...

Research

JoVE Journal

Neuroscience

3.4K ビュー.  Max Planck Institute for Biological Cybernetics. 動物の脳機能マッピングは、リアルタイムの機能的磁気共鳴画像法(fMRI)実験セットアップの恩恵を受けることができます。動物MRIシステムに実装された最新のソフトウェアを使用して、小動物fMRIのリアルタイムモニタリングプラットフォームを確立しました。

ビデオ: Real-Time fMRI Brain Mapping in Animals

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

齧歯類の脳における同時fMRIと電気生理学

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

神経科学

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

デフォルトモードのネットワークを検査する経頭蓋磁気刺激とfMRIを組み合わせ

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

Recording Human Electrocorticographic (ECoG) Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), magnetoencephalography or functional magnetic-resonance imaging. These techniques have either inherently low temporal or low spatial resolution, and suffer from low signal-to-noise ratio and/or poor high-frequency sensitivity. Thus, they are suboptimal for exploring the short-lived spatio-temporal dynamics of many of the underlying brain processes. In contrast, the invasive technique of electrocorticography (ECoG) provides brain signals that have an exceptionally high signal-to-noise ratio, less susceptibility to artifacts than EEG, and a high spatial and temporal resolution (i.e., &lt;1 cm/&lt;1 millisecond, respectively). ECoG involves measurement of electrical brain signals using electrodes that are implanted subdurally on the surface of the brain. Recent studies have shown that ECoG amplitudes in certain frequency bands carry substantial information about task-related activity, such as motor execution and planning1, auditory processing2 and visual-spatial attention3. Most of this information is captured in the high gamma range (around 70-110 Hz). Thus, gamma activity has been proposed as a robust and general indicator of local cortical function1-5. ECoG can also reveal functional connectivity and resolve finer task-related spatial-temporal dynamics, thereby advancing our understanding of large-scale cortical processes. It has especially proven useful for advancing brain-computer interfacing (BCI) technology for decoding a user's intentions to enhance or improve communication6 and control7. Nevertheless, human ECoG data are often hard to obtain because of the risks and limitations of the invasive procedures involved, and the need to record within the constraints of clinical settings. Still, clinical monitoring to localize epileptic foci offers a unique and valuable opportunity to collect human ECoG data. We describe our methods for collecting recording ECoG, and demonstrate how to use these signals for important real-time applications such as clinical mapping and brain-computer interfacing. Our example uses the BCI2000 software platform8,9 and the SIGFRIED10 method, an application for real-time mapping of brain functions. This procedure yields information that clinicians can subsequently use to guide the complex and laborious process of functional mapping by electrical stimulation.
Prerequisites and Planning:
Patients with drug-resistant partial epilepsy may be candidates for resective surgery of an epileptic focus to minimize the frequency of seizures. Prior to resection, the patients undergo monitoring using subdural electrodes for two purposes: first, to localize the epileptic focus, and second, to identify nearby critical brain areas (i.e., eloquent cortex) where resection could result in long-term functional deficits. To implant electrodes, a craniotomy is performed to open the skull. Then, electrode grids and/or strips are placed on the cortex, usually beneath the dura. A typical grid has a set of 8 x 8 platinum-iridium electrodes of 4 mm diameter (2.3 mm exposed surface) embedded in silicon with an inter-electrode distance of 1cm. A strip typically contains 4 or 6 such electrodes in a single line. The locations for these grids/strips are planned by a team of neurologists and neurosurgeons, and are based on previous EEG monitoring, on a structural MRI of the patient's brain, and on relevant factors of the patient's history. Continuous recording over a period of 5-12 days serves to localize epileptic foci, and electrical stimulation via the implanted electrodes allows clinicians to map eloquent cortex. At the end of the monitoring period, explantation of the electrodes and therapeutic resection are performed together in one procedure.
In addition to its primary clinical purpose, invasive monitoring also provides a unique opportunity to acquire human ECoG data for neuroscientific research. The decision to include a prospective patient in the research is based on the planned location of their electrodes, on the patient's performance scores on neuropsychological assessments, and on their informed consent, which is predicated on their understanding that participation in research is optional and is not related to their treatment. As with all research involving human subjects, the research protocol must be approved by the hospital's institutional review board. The decision to perform individual experimental tasks is made day-by-day, and is contingent on the patient's endurance and willingness to participate. Some or all of the experiments may be prevented by problems with the clinical state of the patient, such as post-operative facial swelling, temporary aphasia, frequent seizures, post-ictal fatigue and confusion, and more general pain or discomfort.
At the Epilepsy Monitoring Unit at Albany Medical Center in Albany, New York, clinical monitoring is implemented around the clock using a 192-channel Nihon-Kohden Neurofax monitoring system. Research recordings are made in collaboration with the Wadsworth Center of the New York State Department of Health in Albany. Signals from the ECoG electrodes are fed simultaneously to the research and the clinical systems via splitter connectors. To ensure that the clinical and research systems do not interfere with each other, the two systems typically use separate grounds. In fact, an epidural strip of electrodes is sometimes implanted to provide a ground for the clinical system. Whether research or clinical recording system, the grounding electrode is chosen to be distant from the predicted epileptic focus and from cortical areas of interest for the research. Our research system consists of eight synchronized 16-channel g.USBamp amplifier/digitizer units (g.tec, Graz, Austria). These were chosen because they are safety-rated and FDA-approved for invasive recordings, they have a very low noise-floor in the high-frequency range in which the signals of interest are found, and they come with an SDK that allows them to be integrated with custom-written research software. In order to capture the high-gamma signal accurately, we acquire signals at 1200Hz sampling rate-considerably higher than that of the typical EEG experiment or that of many clinical monitoring systems. A built-in low-pass filter automatically prevents aliasing of signals higher than the digitizer can capture. The patient's eye gaze is tracked using a monitor with a built-in Tobii T-60 eye-tracking system (Tobii Tech., Stockholm, Sweden). Additional accessories such as joystick, bluetooth Wiimote (Nintendo Co.), data-glove (5th Dimension Technologies), keyboard, microphone, headphones, or video camera are connected depending on the requirements of the particular experiment.
Data collection, stimulus presentation, synchronization with the different input/output accessories, and real-time analysis and visualization are accomplished using our BCI2000 software8,9. BCI2000 is a freely available general-purpose software system for real-time biosignal data acquisition, processing and feedback. It includes an array of pre-built modules that can be flexibly configured for many different purposes, and that can be extended by researchers' own code in C++, MATLAB or Python. BCI2000 consists of four modules that communicate with each other via a network-capable protocol: a Source module that handles the acquisition of brain signals from one of 19 different hardware systems from different manufacturers; a Signal Processing module that extracts relevant ECoG features and translates them into output signals; an Application module that delivers stimuli and feedback to the subject; and the Operator module that provides a graphical interface to the investigator.
A number of different experiments may be conducted with any given patient. The priority of experiments will be determined by the location of the particular patient's electrodes. However, we usually begin our experimentation using the SIGFRIED (SIGnal modeling For Realtime Identification and Event Detection) mapping method, which detects and displays significant task-related activity in real time. The resulting functional map allows us to further tailor subsequent experimental protocols and may also prove as a useful starting point for traditional mapping by electrocortical stimulation (ECS).
Although ECS mapping remains the gold standard for predicting the clinical outcome of resection, the process of ECS mapping is time consuming and also has other problems, such as after-discharges or seizures. Thus, a passive functional mapping technique may prove valuable in providing an initial estimate of the locus of eloquent cortex, which may then be confirmed and refined by ECS. The results from our passive SIGFRIED mapping technique have been shown to exhibit substantial concurrence with the results derived using ECS mapping10.
The protocol described in this paper establishes a general methodology for gathering human ECoG data, before proceeding to illustrate how experiments can be initiated using the BCI2000 software platform. Finally, as a specific example, we describe how to perform passive functional mapping using the BCI2000-based SIGFRIED system.

Recording Human Electrocorticographic ECoG Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

We present a method for collecting electrocorticographic signals for research purposes from humans who are undergoing invasive epilepsy monitoring. We show how to use the BCI2000 software platform for data collection, signal processing and stimulus presentation. Specifically, we demonstrate SIGFRIED, a BCI2000-based tool for real-time functional brain mapping.

神経科学の研究とリアルタイム機能皮質マッピングのためのヒトElectrocorticographic（ECOG）信号を記録

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

小動物の慢性神経録音用超軽量電動マイクロドライブの設計およびアセンブリ

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

ハイフィールドのfMRIを使って大人の猫における聴覚皮質の機能イメージング

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

げっ歯類での同時のfMRIと脳深部刺激

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

アン<em&gt;エクスビボ</em&gt;レーザー誘起脊髄損傷モデルがリアルタイムで軸索変性のメカニズムを評価するために

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

管理のリアルタイム fMRI ニューロフィード バック トレーニングのプロトコル

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

脳の細胞外空間の体積分率とねじれを定量するためのテトラメチルアンモニウムによるリアルタイムイオントフォレシス

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

生細胞測定の嗅覚受容体活性化アッセイを用いた、リアルタイム キャンプ

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...