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><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-&amp;beta;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>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>&amp;mu;-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

Universidad de Cartagena UDEC

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

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

ニューロンの光刺激のために手ごろな価格のLEDアレイを使用する

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

神経科学

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性のためのアダルト生まれの嗅覚ニューロンの選択的なウイルス形質導入 in vivoで Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

脳組織の慢性Optogenetic刺激のための光ファイバ注入

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

個々の錐体細胞のハイフィデリティOptogenetic制御法<em&gt;インビボ</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into glass capillaries, pulled to a fine taper, and then sealed using an epoxy to create electrode materials that are used for fast scan cyclic voltammetry testing. The use of bare CFMEs has several limitations, though. First and foremost, the carbon fiber contains mostly basal plane carbon, which has a relatively low surface area and yields lower sensitivities than other nanomaterials. Furthermore, the graphitic carbon is limited by its temporal resolution, and its relatively low conductivity. Lastly, neurochemicals and macromolecules have been known to foul at the surface of carbon electrodes where they form non-conductive polymers that block further neurotransmitter adsorption. For this study, we modify CFMEs with gold nanoparticles to enhance neurochemical testing with fast scan cyclic voltammetry. Au3+ was electrodeposited or dipcoated from a colloidal solution onto the surface of CFMEs. Since gold is a stable and relatively inert metal, it is an ideal electrode material for analytical measurements of neurochemicals. Gold nanoparticle modified (AuNP-CFMEs) had a stability to dopamine response for over 4 h. Moreover, AuNP-CFMEs exhibit an increased sensitivity (higher peak oxidative current of the cyclic voltammograms) and faster electron transfer kinetics (lower &#916;EP or peak separation) than bare unmodified CFMEs. The development of AuNP-CFMEs provides the creation of novel electrochemical sensors for detecting fast changes in dopamine concentration and other neurochemicals at lower limits of detection. This work has vast applications for the enhancement of neurochemical measurements. The generation of gold nanoparticle modified CFMEs will be vitally important for the development of novel electrode sensors to detect neurotransmitters in vivo in rodent and other models to study neurochemical effects of drug abuse, depression, stroke, ischemia, and other behavioral and disease states.

In this study, we modify carbon-fiber microelectrodes with gold nanoparticles to enhance the sensitivity of neurotransmitter detection.

強化された神経化学的検出のための金ナノ粒子修飾炭素繊維マイクロ電極

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into ...

化学

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

ゴールドナノロッドを含む細胞車の作製と光音響分析

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

生物工学

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

生細胞共役ポリマーにより電気的活動の光制御

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

齧歯類の中枢神経系の生体光遺伝学的刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

聴覚神経の光遺伝学的刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

神経細胞の金ナノロッド補助光刺激

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