著者は、部分的に撮影中に彼女の助けのためにシェフィールドさんとJaimeeメインの大学でこの研究を主催したための旅行資金援助と教授ジョン·ヘイコック用NanoVenturesオーストラリアを承認したいと思います。

1.金のNRの準備注：金のNRはレシピ16の数によって合成、または商業ベンダーから購入することができます。 <ol><li> UV-Vis分光法を介して、0.5ナノメートルの解像度で300から1000nmまでの吸収値を記録することによって、金NR溶液の初期光学密度（OD）を測定する。利用可能なキュベットで使用される溶液の体積を変化させる。 </li><li>適切な技術17（ 例えば、UV-Vis分光法、単一粒子誘導結合プラズマ質量分析法、透過型電子顕微鏡）による最初のNPモル濃度を評価するか、ベンダーによって提供される濃度値を使用する。 </li><li>最良の再現性についてはOD = 1に到達するために、初期のAu NRサンプルを希釈する5 mlのストック溶液を調製し、試験した試料のすべてについてODを一定に保つ。希釈溶媒の組成のための商業ベンダーのプロトコルに従ってください。よくわからなければ、脱イオン水を使用しています。 </li><li>二回15分間のAu NR溶液1mlを7800×gで遠心分離した溶液からの任意の化学的過剰を除去する。遠心分離サイクルは、力と時間（ 例えば 20分で5600×g）が18に変化することができる。 </li><li>上清を除去し、脱イオン水でのNRを再懸濁。再懸濁粒子が溶液中で凝集体を形成するかもしれないので、最良の結果を持っているために毎日それらを準備。また、1週間以上、もはや用の冷蔵庫で保管してください。凍結しないでください。 </li><li>細胞培養条件下で使用する前に、5分間のAu NR液を超音波処理した後、（UV照射強度以上400mWのがmに∙-2 254nmで）30分間、UV光で滅菌する。 </li></ol> 2. NG108-15神経細胞ラインの文化と分化<ol><li>細胞培養培地は、無菌のダルベッコ改変イーグル培地（DMEM）を500mlの10％（w / v）のウシ胎児血清（FCS）、L-グルタミン（w / vの）1％、1％の準備（w / v）のペニシリン/ストレプトマイシンおよび0.5％（w / v）のアンホテリシンB 注：サプリメントは、-20℃で保存し、等分し、必要な日に培地に添加することができる。細胞培養培地は、1ヶ月の最大のために無菌状態で冷蔵することができる。 </li><li>細胞分化培地は、1％を含む滅菌DMEMを50mlを調製L-グルタミン、1％（w / v）のペニシリン/ストレプトマイシンおよび0.5％（w / v）のアンホテリシンB（w / v）の</li><li> （37℃、5％CO 2）で加湿雰囲気のインキュベーター中でポリスチレン製のT75フラスコに細胞培養培地10ml中NG108-15神経細胞を成長させる。通常は、3〜4日で準備ができて、各フラスコに1.5〜2×10 5細胞を播種。 2日毎に細胞培養培地を変える。 注：遺伝的ドリフトまたは変動を防ぐために、実験のために通路21より古い細胞を使用しないでください。 </li><li>培養液中の場合には70〜80％コンフルエント、暖かい新鮮な細胞培養培地に培地を変更します。機械的に優しく石油公社によって細胞を切り離す王コンフルエントのフラスコの底に。トリプシンを使用しないでください。 </li><li>遠心分離機600×gで5分間、細胞懸濁液と温かい細胞分化培地2mlで再懸濁細胞ペレット。 </li><li>種子細胞分化培地200μlで組織培養ポリスチレン96ウェルプレート中で2×10 4細胞/ cm 2。 5％CO 2/37℃で1日の実験をインキュベートする。 </li><li> 3.2×10 9の間で-4.2×1 0 10日2上のAu NR液の粒子/ mlを追加し、さらに24時間のためにそれをインキュベートする。対照実験用粒子を追加しないでください。 注：別の対照として、高分化型のピーク吸収波長のAu NPは、比較目的の14のために使用することができる。より一貫性の細胞の挙動については、一定の細胞密度を維持し、細胞播種前のウェル表面を変更しないでください。 </li></ol> 3.神経突起伸長強化<ol><li>カップルシングルモード光ファイバ（開口数= 0.13）を有するレーザとファイバコネクタ（FCコネクタが便利で一般に入手可能である）でそれを終了する。標準的な電力計で出力レーザパワーを測定する。最も効果的な結果を得るために、NRののプラズモン共鳴ピークのレーザーのピーク波長に一致する。 </li><li> NRのインキュベーション後の3日目に、ウェルにFCコネクタを修正。異なるレーザパワーのために、連続波で1分間RTでサンプルとコントロールを照射する。培養は、5％CO 2/37℃で3追加日間進行できるようにします。 3つの独立した測定値の最低レーザー照射を繰り返します。 注：異なる照射時間とパルス周波数は、用途に応じて、選択することができる。 </li><li> （W∙cm -2で）ビーム径とレーザ放射の観点からレーザーを特徴付ける。 <ol><li>標準のシングルモードファイバの場合は、NA = N∙罪θを使用するnが使用されている媒体の屈折率であり、θは 、ファイバを出る光の円錐の半角である（ 図1Aを参照）。 rはビーム半径である三角法で、r =日焼けθ∙d及びdからの繊維とサンプルとの間の距離である。 FCコネクタは、したがって、距離dにおけるサンプル= 2.70±0.20ミリメートルを照らす、ウェルの直径と一致する。ライトは、r =日焼け（SIN-1（NA / n）を ）を与える、水に繊維を抜けた（n = 1.33）dは ∙。 </li><li>後者の式を使用して、ターゲットにレーザービーム半径、対応するビーム面積と平均レーザー放射照度（ビーム面積で割ったレーザパワー）を計算する（ 図1Bの例のグラフを参照）。これらの値は、照射された領域での平均放射照度を表す。 </li><li>エラー伝播の一般理論と測定のためのエラーを評価します。 注：距離between FCコネクタおよびサンプルは、適切なソフトウェア（ImageJの）を使用して、画像の実験装置と後処理の写真により測定することができる。 </li></ol></li><li> 5日目に、実験から細胞分化培地を除去し、3.7％10分間（v / v）のホルムアルデヒド溶液で試料を固定し、その後0.1％20分間（v / v）のトリトンX-100で細胞を透過。 </li><li>結合部位は、未反応のタンパク質をブロックするために60分間サンプルに3％（w / v）のウシ血清アルブミン（BSA）を加える。 4℃で（PBS中5μg/ mlのBSAの1％を補足した）抗βIIIチューブリン一晩のための試料にラベルを付けます。 </li><li> PBS中の1％BSAで0.4〜2μg/ mlの濃度を使用して（ 例えば、TRITC結合抗マウスIgG抗体）は、適切な二次抗体を用いて、暗所で90分間、細胞をインキュベートする。 10分間DAPI（0.1 / mlの中で脱イオン水）で細胞核にラベルを付けます。 （注）抗体共役および濃度MIGhtは、会社のプロトコルに従って変化する。各染色段階後5分間二回PBSで洗浄サンプル。 <img alt="図1" src="/files/ftp_upload/52566/52566fig1.jpg" /></li><li>少なくとも20×対物レンズを使用して落射蛍光または共焦点顕微鏡による画像サンプル。顕微鏡を選択して、二次抗体に応じてフィルタリングします。 DAPIフィルターを選択（λEX = 358 nmのを、λEMは 488 nmの=）を細胞核を可視化する。 </li><li>評価することにより、画像を分析した：i）最大の神経突起の長さ（レコード細胞体の最初に神経突起の先端からの長さ）と、ii）神経細胞あたりの神経突起の数（）は、細胞当たりの神経突起のすべてを総括およびiii）神経突起を有する細胞のパーセンテージ（陽性に染色された核を有する細胞の総数）19によってβIIIチューブリンを発現する細胞の総数を分割する。 </li></ol> 4.細胞内Ca 2+イメージングをレーザ誘起<ol&gt; <li>無水ジメチルスルホキシド（DMSO）10ml中の溶質2gを溶解することによりプルロニックF-127の20％（w / v）のストック溶液を調製する。溶解性を増加させるために、約20分間40℃でプルロニックF-127の溶液を加熱する。 RTで保存した場合、事前に準備をします。 </li><li> NRインキュベーション後3日目に、平衡塩類溶液（BSS; 135ミリモルのNaCl、4.5のKCl、1.5のCaCl 2、0.5のMgCl 2、5.6 mMグルコース、および10mM HEPES、pH7.4）で調製し、5でそれを補ったDMSO中のFluo-4 AMおよび0.1％のμM（w / v）のプルロニックF-127の溶液。 注：BSS溶液を予め調製し、1ヶ月の最大冷蔵することができる。実験の日にサプリメント（のFluo-4 AMとプルロニックF-127）を追加します。 </li><li>細胞分化培地を除去し、補足しBSSソリューションと交換してください。倒立共焦点顕微鏡で最良の結果を得るためには、ウェルのマイクロスライドで細胞を培養する。 </li><li>負荷のFluo-4 AMとNG108-15細胞5％CO 2/37℃、暗所で30分間インキュベートした。インキュベーション時間の後、すべての細胞外のアンロードされた蛍光団を削除するには、BSSで二回のサンプルを洗う。 </li><li>カップルレーザーシングルモード光ファイバとし、標準的な技術を用いて、先端を切断する。 （ すなわち先端が顕微鏡検査の際に繊維軸に垂直と平らにしてください）高品質な表面を確保するために、光学顕微鏡下で生じた先端を観察します。 <ol><li>標準的な電力計で出力レーザパワーを測定する。光ファイバホルダに光送出ファイバを挿入し、マイクロポジショナーにそれを貼り付けます。光ファイバの準備とポジショニングの詳細については、Brown ら 20を参照してください。 注意：（ たとえば、他のラボのユーザーに迷光の露出を防ぐ、取り扱い時にレーザー安全ゴーグルを着用し、レーザー光を直視しないでください）レーザパワーの測定中にレーザーの安全のための一般的なルールに従ってください&lt;。/ LI&gt; </ol></li><li>光変調を監視するために携帯用のレーザーにオシロスコープを接続します。可変周波数（0.5〜2ヘルツ）とパルス長（20〜100ミリ秒）でバイナリ信号を使用してください。 Paviolo ら 13に示す設定に従って、顕微鏡用トランジスタ-トランジスタロジック（TTL）などの変調信号を用いてレーザ入力を接続する。 <ol><li>倒立共焦点顕微鏡下で細胞を置き、250±50ミクロン離れて透過照明モードでの標的細胞からの光送出ファイバを配置。この距離では、ターゲットでのビーム半径を計算するために3.3.1で提示の式を使用しています。 </li></ol></li><li>内部化のFluo-4 AM色素を励起するアルゴンイオンレーザー（488 nm）を使用します（λEX = 494nmで、λEMは 516ナノメートル=）とエンドサイトーシスのNRの励起のための同期化されたLDを。時系列スキャンを収集することにより、少なくとも40倍の対物レンズを用いて試料および対照のRTでの撮像を行う256×256ピクセル/少なくとも4ヘルツの周波数で往復モードのフレームの解像度を持つ。任意のアルゴンイオンレーザー干渉（ベースラインノイズ）を識別するための、LD励起せずに記録を行う。 </li><li>適応的に加重処罰最小二乗アルゴリズム21を使用してデータから背景を削除します。時間の関数として誘発されたCa 2+の変化をプロットします。レベルのベースラインノイズ（3σ）の標準偏差の3倍を閾値化することにより画像後処理ソフトウェアを使用して蛍光強度の過渡的なピークを分析する。 </li></ol>

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The authors have no competing interests to disclose.

このプレゼンテーションで概説プロトコルは文化に、差別化と光学的に外因性の吸収を使用して神経細胞を刺激する方法を説明します。 NR特性（ 例えば、寸法、形状、プラズモン共鳴波長および表面化学）及び（ 等波長、パルス長、繰り返し率、等）、レーザー刺激パラメータは、異なる実験的なニーズに合うように変えることができる。細胞の挙動に対する効果は、標準的?...

最近の研究では、水（&gt; 1400 nmの波長）で赤外光の吸収に関連する過渡的な加熱が神経組織1及び2心筋細胞の細胞内カルシウムトランジェントに活動電位を誘導するために使用され得ることを実証した。赤外光の使用は、潜在的な細かい空間分解能、組織と直接接触の欠如、刺激アーチファクトの最小化、および必要性の除去に遺伝的に、神経補綴物における用途のために大きな関心を調達している刺激前に細胞を修正する（光遺伝学で必要に応じて）1。これらの利点の全てにもかかわらず、最近開発されたサーマルモデルは、標的組織/細胞は、複数の刺激部位および/ ​​または高繰返し率は3,4使用されている累積的加熱効果によって影響を受ける可能性があることを示唆した。 これらの課題に対応するため、研究者らは、外因absorを使用する可能性を認識している神経刺激のための繊維が組織内のより局所的な加熱効果を生成する。 Huang らは、遠隔高周波磁場5をHEK 293細胞中で温度感受性TRPV1チャネルを活性化するために、超常磁性フェライトナノ粒子を使用してこの原理を実証した。この技術は、より深い浸透（磁場が組織と比較的弱く対話）を可能にすることができるが、応答は、ほんの数秒の周期ではなく、バイオニックデバイス5に必要なミリ秒の期間にわたって記録した。同様に、ファラらは、インビトロにおいて黒色微粒子ラット皮質ニューロンの電気刺激を実証した。これらは、潜在的により速い反復率6を可能μJの範囲でμsでエネルギーの数百のオーダーのパルス持続時間を使用して刺激における細胞レベルの精度を示した。 外因性の吸収剤の使用はまた、誘導するために適用されているin vitroでの形態学的変化。 Ciofani らは、超音波7により励起された圧電窒化ホウ素ナノチューブを用いた神経細胞の伸長で約40％の増加を示した。同様に、PC12細胞におけるエンドサイトーシスされた酸化鉄ナノ粒子は、鉄酸化物8の細胞接着分子の活性化を、用量依存的に神経分化を促進することが報告されている。 最近では、神経刺激を支援するための外因性吸収材への関心も、金ナノ粒子（金NPS）を使用することに焦点を当ててきた。 AuのNPは、効率的に、プラズモンピークのレーザ光 ​​を吸収すると熱9の形で周囲の環境へと放散する能力を有する。 10 -利用可能な粒子形状のすべての中で、金ナノロッド（金のNR）の光吸収が便利に（NIR、750-1,400の間の波長近赤外線）生体組織の治療の窓に一致する。また、続きで神経刺激のextは、金のNRの使用は比較的良好な生体適合性と表面機能オプション11の広い範囲を提供します。最近の研究は、分化刺激効果は、NG108-15神経細胞12中のAuのNRの連続レーザ曝露後に誘導できることを示した。同様に、細胞内カルシウムトランジェントは、可変周波数およびパルスで変調されたレーザ照射13を長後のAuのNRで培養した神経細胞で記録した。細胞膜の脱分極はまた、らせん神経節ニューロン14の初代培養中のAuのNRの近赤外レーザー照射後に記録した。照射された金のNRとのin vivoでのアプリケーションの最初のは、つい最近実証されている。オムらは彼らのプラズモンピーク時金のNRを露出し、複合神経活動電位（CNAPS）の振幅の6倍に増加し、ラットの坐骨神経における刺激閾値で3倍の減少を記録した。 ENhanced応答はNRプラズモンピーク15の励起に起因する局所的な加熱効果に起因するものであった。 本論文では、金のNRを用いて培養しNG108-15神経細胞におけるレーザー刺激の効果を調査するためのプロトコルが指定されている。これらのメソッドは、標準的な生物学的技術および材料を用いてインビトロで細胞集団を照射する、シンプルでありながら強力な方法を提供します。プロトコルは、安全な操作で再現性のアライメントを可能にするファイバ結合レーザダイオード（LD）に基づいている。 AuのNRサンプル調製およびレーザー照射方法は、さらに、特定の合成および培養プロトコールはそれぞれ、よく知られている場合に、異なる粒子形状および神経細胞培養物に拡張することができる。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Au NR</td>
			<td>Sigma Aldrich</td>
			<td>716812</td>
			<td></td>
		</tr>
		<tr>
			<td>NG108-15</td>
			<td>Sigma Aldrich</td>
			<td>8811230</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma Aldrich</td>
			<td>D6546</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Life Technologies</td>
			<td>10100147</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Life Technologies</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BDH</td>
			<td>T8532</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A2058</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;III-tubulin</td>
			<td>Promega</td>
			<td>G7121</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC-conjugated anti-mouse IgG antibody</td>
			<td>Sigma Aldrich</td>
			<td>T5393</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4 AM</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Invitrogen</td>
			<td>P6867</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis spectrometer</td>
			<td>Varian Medical Systems Inc.</td>
			<td>Cary 50 Bio</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>Eppendorf</td>
			<td>Mini Spin</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic bath</td>
			<td>Unisonics Australia</td>
			<td>FPX 10D</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Kendro</td>
			<td>Hera Cell 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture centrifuge</td>
			<td>Hettich</td>
			<td>Rotofix 32A</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser diode</td>
			<td>Optotech</td>
			<td>780 nm single mode fibre - coupled LD</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fiber</td>
			<td>Thorlabs</td>
			<td>780 HP</td>
			<td></td>
		</tr>
		<tr>
			<td>Power meter</td>
			<td>Coherent</td>
			<td>Laser Check</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>http://rsb.info.nih.gov/ij/index.html</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescent microscope</td>
			<td>Axon Instruments</td>
			<td>ImageX-press 5000A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#956;-slide well</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>Carl Zeiss Microscopy Ltd.</td>
			<td>LSM 510 meta-confocal microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix</td>
			<td>TDS210</td>
			<td></td>
		</tr>
	</tbody>
</table>

gold nanorod-assisted optical stimulation of neuronal cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

ここで説明するプロトコル1,2、および3を用いて、分化刺激効果は、AuのNPと共に培養NG108-15神経細胞で観察されたレーザー後（金NRは、ポリ（スチレンスルホン酸）のAuのNRおよびシリカ被覆金のNRをコーティングさ） 1.25と7.5 Wとのエクスポージャー·cm -2で。 rhodamineB標識金のNRの共焦点画像は、粒子が、インキュベーション12の1日目から内部移行したことを示した。局在化は主...

Watch this Scientific Journal Video about Gold Nanorod-assisted Optical Stimulation of Neuronal Cells at JoVE.com

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

神経細胞の金ナノロッド補助光刺激

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

gold-nanorod-assisted-optical-stimulation-of-neuronal-cells

Research

JoVE Journal

Neuroscience

8.9K ビュー.  Swinburne University of Technology. This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

ビデオ: Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

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 ...

神経科学

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS：脆弱X症候群と自閉症の可塑性のMechasnismを探検するシータバーストプロトコルを使用する

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

NeuroStar TMSデバイス：うつ病の治療薬としてFDA承認されたプロトコルを実施

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

無傷の感覚器官の前駆細胞の生細胞イメージングショウジョウバエ蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

ニューロンに遺伝的にコードされた非天然アミノ酸を用いた神経タンパク質の光制御

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

マイクロ電極アレイにおけるニューロン細胞培養の刺激に対するネットワーク応答の時間依存的増加

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光干渉断層計: マウス網膜神経節細胞In Vivoイメージング

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

慢性的な経頭蓋電気刺激およびラットの皮質内の記録

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

インビトロにおけるマウス胚性幹細胞からの神経細胞分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

血液由来神経細胞のGMフリー生成

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...