Name: gold nanorod-assisted optical stimulation of neuronal cells
Uploaded: 2015-04-27T14:00:00
Description: Watch this Scientific Journal Video about Gold Nanorod-assisted Optical Stimulation of Neuronal Cells at JoVE.com

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?...

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Chem. 280 (6), 4761-4771 (2005).</li><li>Beauvoit, B., Kitai, T., Chance, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+mitochondrial+compartment+to+the+optical+properties+pf+the+rat+liver:+a+theoretical+and+practical+approach.">Contribution of the mitochondrial compartment to the optical properties pf the rat liver: a theoretical and practical approach.</a> Biophys. J. 67 (6), 2501-2510 (1994).</li></ol>

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 ...

Research

JoVE Journal

Neuroscience

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode 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

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

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

Live-cell Imaging of Sensory Organ Precursor 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 ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

グルタミン酸とGABAの高速マイクロイオン導入：シナプス統合を調査するために便利なツール

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

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

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

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

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

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

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

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 ...