NIMH、NARSADとギャツビー財団が提供する資金。

機器のセットアップ実行されているとfMRIの実験の最初のステップは、セットアップと機器をチェックしています。これらの手順は、任意の順序で行うことができます。 信号のコンディショニング - スキャナは電気と磁気シールドルームに位置しています。制御室からスキャナの部屋に行くすべての電気信号は、MR画像のアーチファクトを作成する可能性のある周波数を除去するフィルタパネルを通過。 Airpuff - airpuffデバイスは、一定の圧力と持続時間の制御されたパフを提供します。目に向かうときは、これは嫌悪が、非外傷性である。空気タンクになるが、我々は、家の空気を使用することができる圧縮空気の供給源に圧力レギュレータ（WPI空気圧PicoPump）、。圧力は30 psiに調整されます。タイミングは50ミリ秒のパフを提供するために調整されます。電磁弁は、コンピュータの信号によって制御されます。圧力レギュレータの出力は、6分の1インチのプラスチックチューブ（タイゴン）を通して被験者に配信されます。チューブが長すぎるまたはソレノイドの開口部と圧縮空気は、チューブの端に到着する時間の間に遅延が生じることではないことが重要です。このような理由から、圧力調整器は、スキャナの部屋の中に位置し、制御信号がない画像アーチファクトがないことを保証するために、フィルタパネルを介して供給されています。 ジュース - ジュースディスペンサーは、ジュースのサイズと数を制御するには、プラスチック製のチューブが口に配信低下します。ジュースディスペンサーは、貯水池、コンピュータ制御のソレノイドバルブ、およびサブジェクトにジュースを提供する長い管で構成されています。すべての電子部品は、スキャナの部屋外であるため、MR信号にはアーティファクトがない。ジュースディスペンサー（クリストの楽器が）タンクを充填し、電磁弁を開くことにより、二回リンスする。それは被験者の好みの飲料で満たされている。システムは、ジュースを数滴の結果を対象に配信されているテストパルスのストリームを生成するためにコンピュータを引き起こすプログラムを実行することによって前にスキャンするためにテストされています。被験者は、ジュースが流れていることを確認します。 バーをかむ - ヘッドの動きは、fMRI画像にいくつかの成果物を作成します。この実験で、被験者は反射的な動きを引き起こす可能性がairpuffs、および嚥下が必要なジュースを受け取る。ヘッドの動きを最小限に抑えるために、bitebarは頭部を安定させるために使用されます。 bitebarは、RFのヘッドのコイルに接続されています。各被験者は、熱可塑性材料から作られたカスタムフィットマウスピースを持っています。 パルスオキシメータ - FMRI対策血液の酸素化（BOLD）。脳内の血液の酸素化は、神経活動によって影響を受ける可能性があります、でもそれは、ハートビートによって影響を受けることができます。心周期に関係する酸素の変化を測定するために、我々は、赤外線センサーを使って指先で血液の酸素化を測定するパルスオキシメータを使用してください。パルスオキシメータは、MR互換性があり、その出力信号は、それをデジタル化し、コンピュータに格納されている制御室へのフィルタパネルを介して供給される。 呼吸ガスモニター - 血液の酸素化はまた、呼吸によって影響を受けることができます。我々は、期限切れのCO2濃度を測定する呼吸ガスモニターで呼吸を測定する。 RGMは、MR互換性があり、その出力信号は、それをデジタル化し、コンピュータに格納されている制御室へのフィルタパネルを介して供給される。 視覚刺激 - 逆投影パネルは被験者の頭の位置に近い穴スキャナの一端に位置しています。被験者は、頭部コイルの上部に取り付けられたミラーを介して背面投影用スクリーンに見える。画像は、スキャナの部屋の外にある液晶プロジェクターによる背面投影スクリーン上に投影されています。プロジェクターからの光ビームはRFシールドホースを介してスキャナの部屋の内部に向けられている。 アイトラッキング - 被写体がいくつかの理由を探している場所を把握することが重要です。眼球運動は、脳内の視覚的な応答に影響を与える網膜、上の視覚刺激の位置に影響を与える。目の動きはまた、被験者が行動反応を示すことの手段として使用することができます。我々は目を追跡するために、&quot;赤外線ビデオ眼球運動記録法&quot;と呼ばれるメソッドを使用します。この方法では、赤外線カメラは、瞳孔を追跡するために使用されます。赤外線エミッタとカメラを特別に設計されたゴーグルに組み込まれています。これらは、視覚刺激を提供する同じゴーグルです。ゴーグルからのデータは、水平​​と垂直眼球位置のためのアナログ信号に目の画像を変換する専用のコンピュータによって処理されます。 主題の準備ドキュメンテーションと同意＆ndash、スキャンする前に、各被験者は、安全性と同意に関して、いくつかのフォームに記入する必要があります。 実験計画シート - プランシートは実験的なプロトコルとパルスシーケンスと、それらが実行される順序を指定します。 物理的な準備は - 被写体がスキャンされる準備ができているとき、彼らが最初に行うことの一つは、騒々しいと高周波スキャナのノイズから自分の聴覚を保護するために耳栓を入れています。彼らはまた、スキャナの被験者との両方と通信するためにMR -互換性のあるヘッドホンを置くと、スキャンセッション中に指示やフィードバックを提供する。 被験者は、ジュースの配信と呼吸ガスモニタリングのための1 / 16インチの行を含む彼らの代弁者、に置きます。 被験者は視覚刺激と眼球運動の監視に使用されるMR互換性のあるゴーグルにして調整置きます。 被験者はスキャナのベッドの上にあり、RFコイルは、彼らの頭の上に配置される。マウスピース（bitebar）はヘッドの移動を防止するためのRFコイルに接続されています。 airpuffチューブ（1 / 6インチタイゴン）は、フレキシブルチューブ（LOC -ライン）を使用して1つ目の近くに位置している。 パルスオキシメータのフィンガークリップを1つの指に取り付け、テープで固定されています。 走査法パルスシーケンス： 構造：我々は被験者の脳の形態の明確な定義を与えるT1強調画像を（構造的な画像）を使用します。被験者は受動的に位置し、約10分間続くこのフェーズでとしても、可能な限り残ります。 機能性：私たちは、神経活動と相関している血液の酸素化の変化を示すためにT2強調EPIシーケンスを（機能的な画像）を使用します。それは主題がジュース、airpuff、および聴覚刺激にさらされていることをこの段階でです。 接続性：私たちは、脳領域間の構造的な接続性を決定するために拡散強調画像（DWI）を使用する。被験者は、単にこの段階で動かずに位置しています。

functional imaging with reinforcement, eyetracking, and physiological monitoring

我々は、意思決定の基礎となる神経回路を研究する脳機能イメージング（fMRI）を使用してください。結果は、意思決定のプロセスをどのように影響するかを理解するために、単純な知覚タスクは欲求と嫌悪補強と組み合わされる。しかし、このようなジュースやairpuffsとしての強化子の使用は、fMRIの課題を作成することができます。補強剤の配信は、fMRIの信号でアーティファクトを作成するヘッドの移動が発生する可能性があります。補強は、また自律神経経路によって媒介される心拍数と呼吸の変化につながることができます。心拍数と呼吸の変化は、直接脳のfMRIの（BOLD）信号に影響を与える可能性がありますし、神経活動に起因する信号の変化に混乱することができます。本発表では、我々は頭部を安定させるための、およびパルスと呼吸を測定するために、制御された方法での強化子を管理するためのメソッドを示しています。

Watch this Scientific Journal Video about Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring at JoVE.com

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

このプレゼンテーションでは、意思決定の基礎となる神経回路を研究するfMRIの使用方法を示します。単純な知覚タスクは成果が意思決定プロセスにどのような影響を与えるか調査する欲求と嫌悪補強と組み合わされる。

強化、Eyetracking、および生体モニタリングによる機能イメージング

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

functional-imaging-with-reinforcement-eyetracking-physiological

Research

JoVE Journal

Biology

11.3K ビュー.  Columbia University. このプレゼンテーションでは、意思決定の基礎となる神経回路を研究するfMRIの使用方法を示します。単純な知覚タスクは成果が意思決定プロセスにどのような影響を与えるか調査する欲求と嫌悪補強と組み合わされる。

ビデオ: Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

タイムラプス顕微鏡で分解アクチンの監視

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

光イメージングによる非侵襲的検出のための蛍光色素で標識幹細胞

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiaeの定量的プロテオミクスとタンパク質の複合体の同定

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

幼虫のモニタリングの心機能キイロショウジョウバエ生理学的研究のための

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

甲殻類との長期致死毒性試験アルテミアfranciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

機能イメージングによるヒト聴覚皮質上のシータバースト刺激の後遺症のマッピング

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

FDG micro-PET/CTマウスにおける褐色脂肪の機能イメージング

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

マウス心筋細胞の単離と生理解析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Concurrent EEG and Functional MRI Recording and Integration Analysis for Dynamic Cortical Activity Imaging

Electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) are two of the fundamental noninvasive methods for identifying brain activity. Multimodal methods have sought to combine the high temporal resolution of EEG with the spatial precision of fMRI, but the complexity of this approach is currently in need of improvement. The protocol presented here describes the recently developed spatiotemporal fMRI-constrained EEG source imaging method, which seeks to rectify source biases and improve EEG-fMRI source localization through the dynamic recruitment of fMRI sub-regions. The process begins with the collection of multimodal data from concurrent EEG and fMRI scans, the generation of 3D cortical models, and independent EEG and fMRI processing. The processed fMRI activation maps are then split into multiple priors, according to their location and surrounding area. These are taken as priors in a two-level hierarchical Bayesian algorithm for EEG source localization. For each window of interest (defined by the operator), specific segments of the fMRI activation map will be identified as active to optimize a parameter known as model evidence. These will be used as soft constraints on the identified cortical activity, increasing the specificity of the multimodal imaging method by reducing cross-talk and avoiding erroneous activity in other conditionally active fMRI regions. The method generates cortical maps of activity and time-courses, which may be taken as final results, or used as a basis for further analyses (analyses of correlation, causation, etc.) While the method is somewhat limited by its modalities (it will not find EEG-invisible sources), it is broadly compatible with most major processing software, and is suitable for most neuroimaging studies.

An EEG-fMRI multimodal imaging method, known as the spatiotemporal fMRI-constrained EEG source imaging method, is described here. The presented method employs conditionally-active fMRI sub-maps, or priors, to guide EEG source localization in a manner that improves spatial specificity and limits erroneous results.