この作品は、ロナルドG.トンプキンス（A.アリアTzika、取締役に衛生研究所（NIH）の助成金ローレンスG. Rahme、衛生研究所（NIH）センターグラント（P50GM021700）の国立研究所研究所のAI063433の国立研究所によって部分的にサポートされていましたA.アリアTzikaの子供の研究助成金（＃8893）のためのNMRのコア）、およびシュリナーの病院。我々はDionyssios Mintzopoulos博士に感謝するこのプロトコルとOvidiu C. Andronesi博士号を開発の初期段階における支援のためのTOBSYパルスシーケンスの支援のための。

パート1：HRMAS測定のための準備ショウジョウバエ <ol><li>標準flylab手順1を使用して 、3日間、新たにeclosingハエを収集し、新鮮なフライ食品を含むバイアルを飛ぶためにそれらを転送する。ハエが直前に実験に5-8日の古いとなるように5日間に収集ハエをインキュベートする。唯一の性別は、（通常は男性）の実験に使用されます。 </li><li>健康的な使用、無傷の野生型は、例えば外傷または遺伝的にハエ1を変更扱わと比較して飛ぶ。 </li><li>実験に先立って、フライ食品24時間の一部（〜0.2ミリリットル）を含む2ミリリットルチューブに単一のハエを置きます。これらの管は、火炎加熱インスリンの注射針を使用して作られたチューブのカップに穴を負うものとします。 </li><li>高精度のバランスを使用してフライの挿入前と直後にチューブの重量を測定し、後者からの最初の値を減算することにより、各フライの重みを定義する。シングル男性のフライは、通常、0.7から1ミリグラムの重量を量る。 </li></ol>パート2：HRMASのローターの準備。 <ol><li>フライの内側に麻酔する分未満の氷の上に単一のチューブを置きます。 </li><li>氷上に置かれたアルミ箔の層の上にフライを置くとハエの傷害を回避しながら、完全なハエの挿入を確実にするために柔らかいブラシを使用してNMRローター挿入の半球状の中空空間にそっとフライを押してください。 </li><li>ローターのローターを挿入し、底部（図1を参照）の間にハエを見つけるために酸化ジルコニウム（ZrO 2）ローターチューブ（直径4mm、50μlの）への挿入を置きます。 </li><li> δトリメチルシリル-プロピオン- 2 ,2,3,3 - D4酸、分子量= 172、：フライとTSP標準溶液（TSPとの間の接触を防ぐために、ネジ付きインサートを閉じて（図1を参照）パラフィルムをかぶせます= 0.00 ppmの、重水- D 2 O、共鳴の化学シフトと定量の両方のリファレンスとして機能する）で50mMの。 </li><li>ローター挿入の上にTSP標準溶液の8μlを添加します。 （図1参照）。 </li><li>固定し（図1参照）トップキャップとローターで設定全体を締めます。 </li></ol>パート3：HRMASデータ収集<ol><li> HRMASプローブにローターを紹介し、マジック角54.7 °（図1参照）に配置します。 </li><li> MASの空気圧ユニットとの組み合わせでBTO - 2000ユニットで4℃で温度を設定します。麻酔を維持するために分光器中にハエを4℃で保存されています。 </li><li> 2 kHzのMASでHRMAS 1 H MRS紡糸速度を設定します。 </li><li> MASのスピードコントローラ（回転周波数）が2.0 ± 0.001 kHzでMASの回転数を安定させます。 </li><li>磁石の準備のための：チューニングと最適なパフォーマンスとシムスペクトルの最適な品質のための磁石のコイルと一致している。 </li><li>にT 2のフィルタとして機能する[N -買収- - （ττ- 180 °）90 °]、カーパーセル- Meiboom -ギル（CPMG）スピンエコーパルスシーケンス2、同期ローターを用いて一次元1 Hスペクトルを取得する全てのサンプルで一次元のデータを取得するために、スペクトルの広がり1 H NMRシングルフライスペクトルを削除します。 MASの回転数（2 kHz）にパルス間の遅延時間（τ=500μsの）を同期する。 32,768（32K）のデータ点で256でトランジェントの数を設定します。アクイジション時間9分。 </li><li>断熱パルスを3でTOBSYシーケンスを使用して、すべてのサンプルの2次元（2D）1 H - 1 H HRMAS NMRシングルフライスペクトルを取得する。買収のパラメータは次のとおりです。2kのデータポイントの直接の次元（11 ppmのスペクトル幅）、1秒リラクゼーションディレイの間に水プレ飽和、インクリメントあたり8スキャン、128ずつ、2秒の総繰り返し時間、45ミリ秒混合時間、および合計獲得29分の時間。 </li></ol>パート4：データ処理/解析<ol><li> MestReCソフトウェアを使用して、標本のMRspectra（Mestrelab研究、www.mestrec.com）を分析する</li><li> 0.5 Hzのライン拡大アポダイゼーション関数を使用し、前にフーリエ変換のすべてのHRMAS 1 HのFIDがに適用されます。 </li><li> δ= 0.0 ppmの（外部標準）でTSPに関連してMRスペクトルを参照する。 </li><li>手動でのスペクトル位相と前のピーク面積の計算にベースラインの広範な成分を減算するホイッタカーのベースラインの推定を適用する。 </li><li> MestReCソフトウェアを使用してピーク面積を推定する。に関する規模のピーク高さはそれぞれ取得したスペクトル（TSPピークの高さ= 1）のためにTSPに。の間に、グループの比率に、グループ内で比較するt検定（両側検定、P &lt;0.05）を使用してください。 </li><li> 2D TOBSYのプロセスパラメータは次のとおりです：両方の次元QSINE = 2窓関数、直接の次元と二次元目、2番目の次元の両方の次元とベースライン補正の位相補正に1kにゼロ充填した2kポイントとFT。 </li><li>スパーキープログラム（TDゴダードとDGネラー、スパーキー3、USCF、使用して2次元スペクトルを得る<a href="http://www.cgl.ucsf.edu/home/sparky/">http://www.cgl.ucsf.edu/</a>ホーム/スパーキー/） </li></ol>パート5：ライブキイロショウジョウバエからの代表的なスペクトルハエ手順は、ライブキイロショウジョウバエのハエから再現性のスペクトルを収集することを許可上記の。野生型（WT）オレゴン- Rで取得した図2と図3に代表的なMRスペクトルが飛ぶ。図2プレゼント1D 1 H HRMAS CPMGスペクトルを。主要な脂質成分[C H 3（0.89 ppm）を、（C H 2）N（1.33 ppm）を、C H 2 C - CO（1.58ppm）、C H 2 C = C（2.02 ppm）を、C H 2 C = O（2.24 ppm）を、C H = C H（5.33 ppm）の]、グリセロール（1,3 - C H 4.10 ppmと4.30 ppmの、2 - CH 2 5.24 ppm）を、と小さな代謝物：β-アラニン（β-アラ、2.57 ppm）を、酢酸（AC、1.97 ppm）を、ホスホコリン（PC、3.22 ppm）とphophoetanolamine（PE、3.23ppm）を検出し、事前の報告4、5に準拠して割り当てられていました。 2.02 ppmのシグナルは、モノ不飽和脂肪酸（すなわち、パルミトレイン）のC H2 - CH = CHの部分のメチレンプロトンに割り当てられていた。不飽和脂肪酸は、- CH = CH -基のプロトンによって生産さ5.33 ppmのシグナルにより同定した。割り当てられていないか、1Dスペクトルを使用して表示されていたことが、小さな代謝物は2次元1 H - 1 Hを用いて検出した TOBSY HRMAS（図3参照）。 <img src="/files/ftp_upload/1710/1710fig1.jpg" alt="図1" /> 図1実験では生体内での設定 重水14.1 T.外部標準トリメチルシリルプロピオン- 2 ,2,3,3 - D4酸（TSP / D 2 O）でのライブショウジョウバエの調査のためのHRMAS 1H MRS。 HRMASプローブでマジック角での回転子位置：正方形で。 <img src="/files/ftp_upload/1710/1710fig2.jpg" alt="図2" /> 図2。ライブキイロショウジョウバエ重量フライの生体 1D HRMAS 1 H CPMGスペクトルで 。脂質成分：C H 3（0.89 ppm）を、（C H 2）N（1.33 ppm）を、C H 2 C - CO（1.58ppm）、酢酸（AC）、C H 2 C = C（2.02 ppm）を、C H 2 C = O（2.24 ppm）を、β-アラニン（β- Ala）を、ホスホコリン（PC）とphophoetanolamine（PE）、グリセロール（1,3 - C H 4.10、4.30 ppmの、2 - C H 2 5.22 ppm）の、C H = C H（5.33 ppm）を。 <img src="/files/ftp_upload/1710/1710fig3.jpg" alt="図3" /> 図3。 生体 2D 1H - 1H の 14.1 T.小型代謝と脂質成分のライブキイロショウジョウバエ重量フライのTOBSY HRMASスペクトルが同定された。代謝産物：アラニン（Ala）、β-アラニン（β- Ala）を、アルギニン（Arg）、グルタミン（Gln）、グルタミン酸（GLU）、PCホスホコリン（PC）、phophoetanolamine（PE）、タウリン（TAU）、α-グルコース（α-グルコース）とグリセリン。脂質成分：C H 3（0.89 ppm）を、（C H 2）N（1.33 ppm）を、C H 2 C - CO（1.58 ppm）を、C H 2 C = C（2.02 ppm）を、C H 2 C = O （2.24 ppm）を、C H = C H（5.33 ppm）を。

<ol>
	<li>Apidianakis, Y., Rahme, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+as+a+model+host+for+studying+Pseudomonas+aeruginosa+infection.">Drosophila melanogaster as a model host for studying Pseudomonas aeruginosa infection.</a> Nat Protoc. 4, 1285-1294 (2009).</li><li>Meiboom, S., Gill, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+spin-echo+method+for+measuring+nuclear+relaxation+time.">Modified spin-echo method for measuring nuclear relaxation time.</a> Rev Sci Instrum. 29, 688-691 (1958).</li><li>Andronesi, O. C., Mintzopoulos, D., Struppe, J., Black, P. M., Tzika, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-state+NMR+adiabatic+TOBSY+sequences+provide+enhanced+sensitivity+for+multidimensional+high-resolution+magic-angle-spinning+1H+MR+spectroscopy.">Solid-state NMR adiabatic TOBSY sequences provide enhanced sensitivity for multidimensional high-resolution magic-angle-spinning 1H MR spectroscopy.</a> J Magn Reson. 193, 251-258 (2008).</li><li>Astrakas, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton+NMR+spectroscopy+shows+lipids+accumulate+in+skeletal+muscle+in+response+to+burn+trauma-induced+apoptosis.">Proton NMR spectroscopy shows lipids accumulate in skeletal muscle in response to burn trauma-induced apoptosis.</a> Faseb J. 19, 1431-1440 (2005).</li><li>Fan, T. W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolite+profiling+by+one-+and+two+dimensional+NMR+analysis+of+complex+mixtures.">Metabolite profiling by one- and two dimensional NMR analysis of complex mixtures.</a> Prog Nuc Magn Reson Spec. 28, 161-219 (1996).</li><li>Null, B., Liu, C. W., Hedehus, M., Conolly, S., Davis, 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=High-resolution,+in+vivo+magnetic+resonance+imaging+of+Drosophila+at+18.8+Tesla.">High-resolution, in vivo magnetic resonance imaging of Drosophila at 18.8 Tesla.</a> PLoS One. 3, e2817-e2817 (2008).</li><li>Zektzer, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+signal+to+noise+in+high-resolution+magic+angle+spinning+total+correlation+spectroscopy+studies+of+prostate+tissues+using+rotor-synchronized+adiabatic+pulses.">Improved signal to noise in high-resolution magic angle spinning total correlation spectroscopy studies of prostate tissues using rotor-synchronized adiabatic pulses.</a> Magn Reson Med. 53, 41-48 (2005).</li></ol>

動物実験は、マサチューセッツ総合病院動物実験の研究レビュー委員会の委員会が定めるガイドラインおよび規則に従って行った。

果実における in vivo MRI での実現可能性の最近の報告を除いて6ハエ、 ショウジョウバエを用いるin vivo MRS研究ではまだ報告されていない。現在のプロトコルでは、我々は生物学的に重要な分子を検出するためのin vivoで HRMAS 1 H - MRSのアプローチで小説の実装について説明します。ゼロフライ死亡率を達成しながら、具体的には、ライブ<e...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Deuterium oxide</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>7789-20-0</td>
<td>&nbsp;</td>
<tr>
<td>3-(trimethylsilyl)propionic-2,2,3,3-d4 acid</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>24493-21-8</td>
<td>&nbsp;</td>
<tr>
<td>agar, sucrose, yeast, cornmeal</td>
<td>Food</td>
<td>Genesee Scientific Corporation</td>
<td>&nbsp;</td>
<td><a href="http://www.flystuff.com/">http://www.flystuff.com/</a></td>
</tr>
<tr>
<td>Oregon RS or Canton-S flies</td>
<td>Adult fly lines</td>
<td>Bloomington Stock Center</td>
<td>&nbsp;</td>
<td><a href="http://flystocks.bio.indiana.edu/">http://flystocks.bio.indiana.edu/</a></td>
</tr>
<tr>
<td>Paintbrush</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>(size 0)</td>
</tr>
<tr>
<td>2ml tubes</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>K749521-1590</td>
<td>&nbsp;</td>
<tr>
<td>Fly incubators</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>high humidity capacity (60-75%), adjustable temperature, and a 12 h:12 h light: dark cycle.</td>
</tr>
<tr>
<td>Bruker Bio-Spin Avance NMR spectrometer (600.13 MHz) 4mm triple resonance (1H, 13C, 2H) HRMAS probe </td>
<td>Equipment</td>
<td>Bruker Bio-Spin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BTO-2000 unit in combination with a MAS pneumatic unit</td>
<td>Equipment</td>
<td>Bruker Bio-Spin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>4mm zirconium oxide rotor (capacity 50 ul)</td>
<td>Equipment</td>
<td>Bruker Bio-Spin</td>
<td>B3829 (Bruker store)</td>
<td>&nbsp;</td>
<tr>
<td>MestReC (Mestrelab Research) </td>
<td>Software</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1D NMR spectra analysis <a href="http://mestrelab.com/">http://mestrelab.com/</a></td>
</tr>
<tr>
<td>SPARKY 3, USCF</td>
<td>Software</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2D NMR spectra
analysis
 <a href="http://www.cgl.ucsf.edu/home/sparky/">http://www.cgl.ucsf.edu/home/sparky/</a></td>
</tr>		</tbody>
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magnetic resonance spectroscopy of live drosophila melanogaster using magic angle spinning

高分解能マジック角スピニング（HRMAS）プロトン磁気共鳴分光法（<sup&gt; 1</sup&gt; H - MRS）のスペクトル線幅を向上させ、抽出、無傷の細胞、細胞培養、および代謝と細胞のプロセス間の関係を調査するため、さらに重要な無傷の組織から得られるため、高分解能スペクトルを可能にする新たな非破壊手法です。<em&gt; in vivoで</em&gt; HRMAS<sup&gt; 1</sup&gt; H - MRSの研究では、ライブショウジョウバエで報告されてこなかった<em&gt;キイロショウジョウバエ。</em&gt;<em&gt;ショウジョウバエ、</em&gt;単純な遺伝的生物として、複数の生物学的機能と様々な進化的に保存されたシグナル伝達経路は、全体の生体レベルで確認することができますし、それは遺伝学と生理学を調査するための有用なモデルです。この目的のために、我々は、開発と実装<em&gt; in vivoで</em&gt; HRMAS<sup&gt; 1</supライブ調査する&gt; H - MRS法<em&gt;ショウジョウバエ</em&gt;ここでは14.1 Tで、我々はHRMASを概説<sup&gt; 1</supの分子特性評価のための&gt; H - MRSプロトコル<em&gt;ショウジョウバエ</em&gt; HRMASプローブを備えた従来のMRスペクトロメータを持つ。この手法は、小説です。<em&gt; in vivoで、</em&gt;非破壊<em&gt;ショウジョウバエ</emしたがって、疾患のバイオマーカーの同定を可能にし、&gt;代謝物測定のアプローチは、新規治療開発に貢献するかもしれない。

Watch this Scientific Journal Video about Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning at JoVE.com

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

この手法は、ライブの分子特性評価のためにプロトンMRスペクトロスコピー（HRMAS 1H - MRS）を回転の高分解能マジック角の使用を可能にする<em&gt;キイロショウジョウバエ</em&gt; HRMASプローブを備えた従来の14.1テスラ分析計と。

ライブの磁気共鳴分光法キイロショウジョウバエマジックアングルスピニングを使用して

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral ...

magnetic-resonance-spectroscopy-live-drosophila-melanogaster-using

Research

JoVE Journal

Neuroscience

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

12.4K ビュー.  Massachusetts General Hospital, Harvard Medical School. この手法は、ライブの分子特性評価のためにプロトンMRスペクトロスコピー（HRMAS 1H - MRS）を回転の高分解能マジック角の使用を可能にするキイロショウジョウバエ HRMASプローブを備えた従来の14.1テスラ分析計と。

ビデオ: Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

複数のマウスの神経解剖学的磁気共鳴イメージング

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

神経科学

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

で逃避行動のOptogenetic刺激<em&gt;キイロショウジョウバエ</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

における自発運動量の測定<em&gt;キイロショウジョウバエ</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

一次運動野代謝に及ぼすバイ半球経頭蓋電気刺激効果の測定のためのツールと​​しての磁気共鳴分光法の利用

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

のライブイメージング<em&gt;ショウジョウバエ</em&gt;幼虫の神経芽細胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

組織抽出物の高分解能核磁気共鳴分光法によるラット脳のメタボローム解析

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

で社会空間に影響を与える条件<em&gt;キイロショウジョウバエ</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

臭気誘導挙動の高分解能定量<em&gt;キイロショウジョウバエ</em&gt;使用<em&gt; Flywalk</em&gt;パラダイム

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

パスの長さのプロトコルを採餌<em&gt;キイロショウジョウバエ</em&gt;幼虫

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

キイロショウジョウバエ幼虫と大人の脳の代謝解析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...