我々 はサンドラ N. Manosalvas-Kjono、プリム Ladpli に感謝、ファラー Memon、Divya Gopisetty、ヴェロニカ ・ サンチェスのサポート デバイス設計、変異動物の世代。この研究はサポートされている NIH 助成金 R01EB006745 (BLP)、(MBG) に R01NS092099、K99NS089942 (MK) に、(ALN) を F31NS100318、受信した欧州連合のホライゾン 2020年研究と技術革新の下で欧州研究会議 (ERC) からの資金調達のプログラム (付与契約号 715243 MK)。

<ol>
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著者が明らかに何もありません。

このプロトコルは、マイクロ流体チップの中に閉じ込め回虫の皮膚に正確な機械的刺激を提供する方法を示します。それは生物的質問に答えるための物理的な刺激の統合を容易にするものです、メカノバイオロジー研究生物ラボでの効率化を目指します。このメソッドは、線虫の機械刺激受容ニューロンの機能を評価するために前の試金を拡張します。以前の定量的、半定量的な手法?...

タッチの感覚は、彼らの環境に関する重要な情報を持つ動物を提供します。適用された力によって、無害な楽しい、または痛みを伴う、タッチは感知されます。タッチの中に組織の変形は、最もよくイオン チャンネル受容体蛋白質を表現する皮膚に埋め込まれた特殊な受容細胞によって検出されます。タッチと痛みの間にイオン チャネル活性化への力覚のリンク手順が完全に理解されていません。さらに少ないは、皮膚組織が機械的変形、メカノレセプターがひずみの変化を検出するかどうかをフィルターか1,2,3のストレスについて知られています。一部、このギャップの理解では、生きている動物の皮の表面に細胞レベルで反応を観察しながら正確な機械的刺激を適用する適切なツールの欠如から発生します。適用し、隔離されたセル4,5で力を測定してもリビングの Piezo1 受容体を有効にして、原子間力顕微鏡を広く使用されているに対し細胞6、特に生きている動物を使用して同様の実験は、 線虫、被写体の本質的なモビリティのために悪名高く挑戦されています。この挑戦は、伝統的寒天パッド1,7,8,9の個々 の動物を固定する獣医または手術用シアノアクリ レート系接着剤を使用して回避します。このアプローチは、生産されているが、接着剤と機械的コンプライアンスの軟寒天培地表面固定化に必要なスキルに関連する制限があります。マイクロ流体戦略は、接着にリンクされている合併症のいくつかを回避する無料の代替です。
線虫は動物のサイズのために、マイクロ流体技術のためのよい適合をある完全にマップされた神経系の遺伝モデル生物です。マイクロ ベースのデバイスは高分解能イメージングと関連する神経調節刺激の配信を行いながらそれ以外の場合非常にモバイルの動物を抑制できるという利点を提供しています。マイクロ流体の助けを借りて技術、動物の生活は害10,11, 生涯12,13上行動活動の監視と高解像度を実現することがなく固定化することができます。神経活動14,15,16,17のイメージング。さらに、タッチと痛みの感覚は、生理1,8、機械式4,18,19、特徴付けることができるために必要な多くの受容ニューロン分子レベル20,21,22。
線虫 c. エレガンス6 TRNs、動物の前部 (ALML/R と AVM) を支配する 3 つのうち、3 つは動物の後部 (PLML/R と PVM) を支配するを使用してその体壁に穏やかな機械的刺激を感覚します。生化学的信号に適用された力の伝達に必要なイオン チャネル分子は TRNs8で幅広く研究されています。本稿では正確な機械力を固定化c. の elegansの皮膚に適用する研究者を可能にするマイクロ流体プラットフォーム23回虫、光イメージングによるその内部組織の変形を読みながら。明確に定義された機械的刺激を提示に加えてカルシウム トランジェントを受容ニューロン細胞レベル下の解像度で記録し、形態学的および解剖学的機能と相関できます。デバイスは、単一の動物を保持し、その皮膚 (図 1および図 2) 六つの空気圧アクチュエータのチャンネルの横にあるを示す中央トラップ チャネルで構成されます。六つのチャンネルは、各ワームの 6 TRNs に機械的刺激を提供するトラップ チャネルに沿って配置されます。これらのチャネルは、薄い PDMS のダイヤフラムがあり、外部の空気圧力源 (図 1) によって駆動することができますでトラップ室から区切られます。圧力に対する変形を校正この記事で測定を提供しています。各アクチュエータは、個別に対処し、好みの受容を促進するために使用できます。ピエゾ駆動圧力ポンプを使用して圧力を配信、任意の代替デバイスを使用できます。TRNs体内をアクティブにし、大人の線虫に機械的刺激を提供する、デバイスに大人動物の読み込み、カルシウム イメージングを実行する適切な操作装置を示す圧力プロトコルを使用できることを示す実験と結果の分析します。デバイスの作製は、2 つの主な手順で構成されています: 1) SU-8; から金型を作るフォトリソグラフィー・ 2) デバイスにする PDMS を成形します。簡潔、明瞭のために以前に公開された記事およびプロトコル24,25金型とデバイスを生成する方法について読者が呼ばれます。

<table>
	<tbody>
		<tr>
			<td>Chrome mask</td>
			<td>Compugraphics (http://www.compugraphics-photomasks.com/)</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>Chrome mask</td>
			<td>Mitani-Micronics (http://www.mitani-micro.co.jp/en/)</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>Chrome mask</td>
			<td>Kuroda-Electric (http://www.kuroda-electric.eu/</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>4&#39;&#39; Silicon wafer (B-test)</td>
			<td>Stanford Nanofabrication Facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2002</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2050</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin-coater</td>
			<td>Laurell Technologies</td>
			<td>WS-400BZ-6NPP/LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>Exposure timer</td>
			<td>Optical Associates, Inc</td>
			<td>OAI 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumination controller</td>
			<td>Optical Associates, Inc</td>
			<td>2105C2</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 developer</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>A426F-1GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A18-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloromethylsilane (TCMS)</td>
			<td>Sigma-Aldrich</td>
			<td>92361-500ML</td>
			<td>Caution: TCMS is toxic and water-reactive</td>
		</tr>
		<tr>
			<td>Sylgard 184 Elastomer Kit</td>
			<td>Dow Corning</td>
			<td></td>
			<td>PDMS prepolymer</td>
		</tr>
		<tr>
			<td>Biopsy punch, 1 mm</td>
			<td>VWR</td>
			<td>95039-090</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen Plasma Asher</td>
			<td>Branson/IPC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small metal tubing (0.635 mm OD, 0.4318 mm ID, 12.7 mm long); gage size 23TW</td>
			<td>New England Small Tube Corporation</td>
			<td>NE-1300-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene syringe filter, 0.22 &#956;m</td>
			<td>Thermo Scientific</td>
			<td>725-2520</td>
			<td>to filter all solution, small particles would clog the chip</td>
		</tr>
		<tr>
			<td>Polyethylene tubing; 0.9652 mm OD, 0.5842 mm ID</td>
			<td>Solomon Scientific</td>
			<td>BPE-T50</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 1 ml</td>
			<td>BD Scientific</td>
			<td>309628</td>
			<td>for worm trapping and release</td>
		</tr>
		<tr>
			<td>Syringe, 20 ml</td>
			<td>BD Scientific</td>
			<td>309661</td>
			<td>for gravity-based flow</td>
		</tr>
		<tr>
			<td>Gilson Minipuls 3, Peristaltic pump</td>
			<td>Gilson</td>
			<td></td>
			<td>to suck solutions and worms out of the chip</td>
		</tr>
		<tr>
			<td>Microfluidic flow controller, equipped with 0&#8211;800 kPa pressure channel</td>
			<td>Elveflow</td>
			<td>OB1 MK3</td>
			<td>pressure delivery</td>
		</tr>
		<tr>
			<td>Water-Resistant Clear Poly- urethane Tubing, 4 mm ID and 6 mm OD</td>
			<td>McMaster-Carr</td>
			<td>5195 T52</td>
			<td>connection from house air to pressure pump</td>
		</tr>
		<tr>
			<td>Water-Resistant Clear Polyurethane Tubing, 2.6mm ID and 4mm OD</td>
			<td>McMaster-Carr</td>
			<td>5195 T51</td>
			<td>connect pressure pump to small tubng</td>
		</tr>
		<tr>
			<td>Push-to-Connect Tube Fitting for Air</td>
			<td>McMaster-Carr</td>
			<td>5111K468</td>
			<td>metric - imperial converter</td>
		</tr>
		<tr>
			<td>Straight Connector for 6 mm &#215; 1/4&#8243; Tube OD</td>
			<td>McMaster-Carr</td>
			<td>5779 K258</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMI 4000 B microscopy system</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>63&#215;/1.32 NA HCX PL APO oil objective</td>
			<td>Leica</td>
			<td>506081</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamamatsu Orca-Flash 4.0LT digital CMOS camera</td>
			<td>Hamamatsu</td>
			<td>C11440-42U</td>
			<td></td>
		</tr>
		<tr>
			<td>Lumencor Spectra X light engine</td>
			<td>Lumencor</td>
			<td></td>
			<td>With cyan and green/yellow light source</td>
		</tr>
		<tr>
			<td>Excitation beam splitter</td>
			<td>Chroma</td>
			<td>59022bs</td>
			<td>in the microscope</td>
		</tr>
		<tr>
			<td>Hamamatsu W-view Gemini Image splitting optics</td>
			<td>Hamamatsu</td>
			<td>A12801-01</td>
			<td>to split green and red emission and project them on different areas on the camera chip</td>
		</tr>
		<tr>
			<td>Emission beam splitter</td>
			<td>Chroma</td>
			<td>T570lpxr</td>
			<td>in the image splitter</td>
		</tr>
		<tr>
			<td>Emission filters GCamp6s</td>
			<td>Chroma</td>
			<td>ET525/50m</td>
			<td>in the image splitter</td>
		</tr>
		<tr>
			<td>Emission filters mCherry</td>
			<td>Chroma</td>
			<td>ET632/60m</td>
			<td>in the image splitter</td>
		</tr>
	</tbody>
</table>

using a microfluidics device for mechanical stimulation and high resolution imaging of c. elegans

1 つの中央目標メカノバイオロジーの蛋白質および細胞のメカニカル ストレスの相互影響を理解することです。その重要性にもかかわらず機械的ストレスの細胞機能に及ぼす影響はまだよくわかっていません。いくつかのツールは、組織や細胞の同時変形、生きている動物の細胞の活動のイメージングと線虫など、それ以外の場合高度なモバイル モデル生物運動の効率的な制限を有効にするためにこの知識のギャップが存在する部分には、線虫。線虫 c. エレガンスのサイズが小さいマイクロ ベースの研究装置に優れた一致のため、マイクロ流体デバイスを用いた固定化のためのソリューションを提示されています。これらのデバイスは高分解能イメージングを使用、動物はポリジメチルシロキサン (PDMS)、ガラス、機械的な力や電気生理学的記録の配信のための物理的なアクセスを制限することに完全に包まれています。最近では、高解像度蛍光顕微鏡と互換性のあるトラップ デザインと空気圧アクチュエータを統合したデバイスを作成しました。作動チャネルは、薄い PDMS ダイヤフラムでワーム トラップ チャネルから区切られます。この振動板は、外部ソースからの圧力を適用することによってワームの側面にそらされます。デバイスは、個々 の機械受容ニューロンをターゲットできます。これらのニューロンの活性化でイメージはカルシウムの遺伝子にコードされた指標と高解像度。この記事は、接触受容体ニューロン (TRNs) でカルシウムに敏感なアクティビティ インジケーター (GCaMP6s) を表現する線虫の系統を用いた一般的な方法を示します。メソッドはしかし、プローブと TRNs にもカルシウム センサーに限定ではない、他の機械的に敏感な細胞またはセンサーに拡張できます。

SU 8 リソグラフィとチップ接合 リソグラフィ プロトコルと PDMS 成形標準的な手順に従います。詳細は23,24,25,26他の場所で見つけることができます。PDMS は硬化後問題なくウェーハをはがします必要があります。PDMS 剥離中に SU 8 機能をはぎ取る、SU 8 接着層?...

Watch this Scientific Journal Video about Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans at JoVE.com

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

メカノバイオロジー研究のための新しいツールはどのように機械的ストレスを理解に必要な生化学的経路を活性化し、生体反応を引き出します。ここでは、細胞応答の高分解能イメージングをできるようにマイクロ トラップで固定化動物の選択的な機械的刺激のための新しい方法を紹介します。

マイクロ流体デバイスを用いた機械的刺激と線虫の高解像度イメージング

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

using-microfluidics-device-for-mechanical-stimulation-high-resolution

Research

JoVE Journal

Neuroscience

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

9.8K ビュー.  Stanford University. メカノバイオロジー研究のための新しいツールはどのように機械的ストレスを理解に必要な生化学的経路を活性化し、生体反応を引き出します。ここでは、細胞応答の高分解能イメージングをできるようにマイクロ トラップで固定化動物の選択的な機械的刺激のための新しい方法を紹介します。

ビデオ: Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

トランスジェニックを用いてアッセイするβ-アミロイドの毒性 C.エレガンスモデル

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

神経科学

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

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

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

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

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

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 ...

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is 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.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

TMSの状態 - 依存性の効果：動機の閃光の挙動を見

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

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 ...

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments in vision research require displays with more than 12 bits of luminance resolution. Several solutions are available. Bit++ 1 and DataPixx 2 use the Digital Visual Interface (DVI) output from graphics cards and high resolution (14 or 16-bit) digital-to-analog converters to drive analog display devices. The VideoSwitcher 3 described here combines analog video signals from the red and blue channels of graphics cards with different weights using a passive resister network 4 and an active circuit to deliver identical video signals to the three channels of color monitors. The method provides an inexpensive way to enable high-resolution monochromatic displays using conventional graphics cards and analog monitors. It can also provide trigger signals that can be used to mark stimulus onsets, making it easy to synchronize visual displays with physiological recordings or response time measurements.
Although computer keyboards and mice are frequently used in measuring response times (RT), the accuracy of these measurements is quite low. The RTbox is a specialized hardware and software solution for accurate RT measurements. Connected to the host computer through a USB connection, the driver of the RTbox is compatible with all conventional operating systems. It uses a microprocessor and high-resolution clock to record the identities and timing of button events, which are buffered until the host computer retrieves them. The recorded button events are not affected by potential timing uncertainties or biases associated with data transmission and processing in the host computer. The asynchronous storage greatly simplifies the design of user programs. Several methods are available to synchronize the clocks of the RTbox and the host computer. The RTbox can also receive external triggers and be used to measure RT with respect to external events. 
Both VideoSwitcher and RTbox are available for users to purchase. The relevant information and many demonstration programs can be found at <a href="http://lobes.usc.edu/">http://lobes.usc.edu/</a>.

Conventional computer hardware can not generate visual stimuli with sufficiently high grayscale resolution and measure response times with sufficient accuracy. We describe how to use the VideoSwitcher to produce high-resolution monochromatic displays, and the RTbox to measure response times with high accuracy on conventional computer hardware.

有効にする高解像度を表示し、従来のコンピュータ上で正確な応答時間の測定値をグレースケール

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

人間の中脳の高解像度機能的磁気共鳴画像法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

開発神​​経上皮における細胞挙動の高解像度のライブイメージング

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

シナプス蛍光の自動定量<em&gt; C.エレガンス</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

Cの生体内神経カルシウムイメージングでエレガンス

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

マイクロ流体デバイスを用いた機械的刺激と線虫の高解像度イメージング

要約

さらに動画を探す

この動画の章

トランスジェニックを用いてアッセイするβ-アミロイドの毒性 C.エレガンスモデル

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

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

TMSの状態 - 依存性の効果：動機の閃光の挙動を見

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

有効にする高解像度を表示し、従来のコンピュータ上で正確な応答時間の測定値をグレースケール

人間の中脳の高解像度機能的磁気共鳴画像法

開発神経上皮における細胞挙動の高解像度のライブイメージング

シナプス蛍光の自動定量

Cの生体内神経カルシウムイメージングでエレガンス

マイクロ流体デバイスを用いた機械的刺激と線虫の高解像度イメージング

要約

さらに動画を探す

この動画の章

トランスジェニックを用いてアッセイするβ-アミロイドの毒性 C.エレガンスモデル

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

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

TMSの状態 - 依存性の効果：動機の閃光の挙動を見

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

有効にする高解像度を表示し、従来のコンピュータ上で正確な応答時間の測定値をグレースケール

人間の中脳の高解像度機能的磁気共鳴画像法

開発神​​経上皮における細胞挙動の高解像度のライブイメージング

シナプス蛍光の自動定量

Cの生体内神経カルシウムイメージングでエレガンス

開発神経上皮における細胞挙動の高解像度のライブイメージング