著者は、彼の継続的な技術サポートのためにIkiel Ptitoに感謝します。 MPにNSERC助成金。

パート1：組織の前処理<ol><li>組織はよくパラホルムアルデヒド、グルタルアルデヒド、またはホルマリンで灌流してください。これは通常、他の臓器を収穫するために使用される標準transcardial灌流によって達成することができます。完全にexsanguinated.Thisが続いされるまで本研究では被験者は深く、（10 mg / kgを、IM）塩酸ケタミンで鎮静したペントバルビタールナトリウムの過剰投与（25 mg / kgを、IV）を使用して、安楽死させ、0.1MのPBSでtranscardially灌流5分（〜1リットル）のためのPBS中4％パラホルムアルデヒド溶液で。 </li></ol>パート2：固定装置ブロッキング<ol><li>前の定位フレームにヘッドを配置するHorsley-Clarke/interaural面のゼロの座標は、注意が必要です。これは耳の間に理論上の中間点です。この平面を測定するには、耳のバーが同じように装置に装着する必要がある、そして定位マニピュレータのアームで手術用メスの刃を配置し、耳の棒の間に中間点を測定する。これは、研究のニーズに基づいて組織をブロックする場所を決定するために重要です。これが完了すると頭蓋骨は、定位固定装置に固定のために準備する必要があります。 </li><li>定位フレームに頭部を置くために下顎は骨Rongeursとメスで削除する必要があります。さらに皮膚、筋肉、および結合組織は、頭蓋骨を露出させるために削除する必要があります。結合組織が頭蓋骨から削除されると、頭蓋冠を（後頭骨の表形式の部分だけでなく、頭頂葉、広告前頭葉の骨）離れ​​てチッピングすることにより、脳を公開。頭蓋冠の本実験の一部ではすべての準備は削除されました。外耳道は、定位フレームに頭部を置くためにそのままにする必要があるので完全に頭の骨を削除しないように注意してください。残りの硬膜を露出脳から削除する必要があります。頭蓋骨は今定位フレームに配置する準備が整いました。 </li><li>一定位手術を行うというのと同じ方法で、頭がしっかり定位固定装置に固定されているような眼、口蓋、およ​​び耳のバーを調整します。所定前方/後方（/ P）の座標に定位マニピュレータを配置し、脳の外側面にマニピュレータを移動する。徐々に完全にして内側に移動し、再度ブレードを下げ、脳からのブレードを高める、脳内にブレードを下げる。ブレードは、反対半球の外側面に達するまで、これらの2つの手順を繰り返します。これは、最初の冠状ブロックが完成。脳全体がブロックされるまで、後続のコロナのブロックに対して/ Pの軸と繰り返しでマニピュレータの1センチメートルを移動する。 </li></ol>パート3：頭蓋骨から脳を削除する<ol><li>定位フレームからヘッドを取り外して、あなたの手のひらの上で露出脳を開催。軽くcoddling手のひらサイズの脳を配置することにより、脳を保護すると、前頭葉全体で頭蓋骨の脳のこの最小限に抑える動きをして小指を配置してください。脳の軟膜表面の乾燥を防ぐために、PBSの一部が脳全体にガーゼを湿らせた置きます。しっかりと脊柱と一緒に頭蓋骨とチップ離れて、残りの後頭部や側頭骨で頭をホールド。これは、脳の基底部と外側の側面を公開します。最後に、嗅球へのアクセスを許可する任意の残りの前頭骨と鼻の骨を、削除してください。あなたが頭蓋底を削除すると、脳がわずかに動くと前頭葉が源泉の骨のギザギザのエッジに損傷を受けることができるので、それは最後の前頭骨を除去することが重要である。切り取ると、残りの硬膜を除去。慎重に、脳と頭蓋骨から無料の脳の下にメスをスライドさせ、脳の前部を持ち上げ。 </li></ol>パート4：測定<ol><li>前に凍結させることができる便利な測定値の数があります。キャリパーと脳の例については、A / P軸。さらに、特定の密度は、体積変位（表1）で重量を割った後、メスシリンダー中の水の変位による体積を測定し、脳の重量を量ることによって測定することができます。 </li></ol>パート5：脳をブロック終了。 <ol><li>手術用メスの刃が脳内に挿入されたときに、通常、それは完全に脳の完全な背腹範囲に浸透するのに十分な長さがありません。脳が削除され、総測定値を取得すると、組織スライス刃を取ると脳の腹側（図2）に至るまで、脳をブロック終了。 </li><li>ブロックを凍結する前に、それは脳のシンクまで4˚Cで維持傾斜PBS -バッファショ糖溶液（10、20、および30％）で組織をcryoprotectする必要があります。それは、通常、容器の底に沈むためにブロックの30％で10％、20％で2〜3日と追加の3-5日で一晩インキュベーションを取ります。番目の日々の変化E 30％ショ糖溶液をお勧めします。 </li></ol>パート6：代表的な結果脳が頭蓋骨から削除されている回だけ行うことができます総形態的測定値の数があります。これらは、/ Pの長さ、重量、および特定の密度を（表1）が含まれます。我々は一般的に1CM（図1）を測定する6〜7ブロックに脳をブロックする。各ピースを撮影される（図2）と、さらなる研究の必要性に応じて解剖したり、段階的なショ糖溶液中で凍結のために調製することができる。 <table border="1"><tbody><tr><td> 対象と </td><td> / Pの長さ （㎜） </td><td> 重さ （グラム） </td><td> 水置換 （ml）を </td><td> 特定の密度 （g / mlの） </td></tr><tr><td> O2303 - 2 - 1 - 1 </td><td> 64.3 </td><td> 28.1 </td><td> 24 </td><td> 1.171 </td></tr><tr><td> O5180 - 1 </td><td> 71.3 </td><td> 38.7 </td><td> 34 </td><td> 1.138 </td></tr><tr><td> O2708 - 3 - 1 </td><td> 62.8 </td><td> 28.7 </td><td> 26 </td><td> 1.104 </td></tr><tr><td> O9184 - 4 - 2 </td><td> 65.3 </td><td> 29.5 </td><td> 24 </td><td> 1.229 </td></tr><tr><td> N459 - 1 - 14 - 2 </td><td> 68.2 </td><td> 31.6 </td><td> 26 </td><td> 1.215 </td></tr><tr><td> AVERAGE </td><td> 66.38 </td><td> 31.32 </td><td> 26.8 </td><td> 1.171 </td></tr><tr><td> STD DEV </td><td> 3.38 </td><td> 4.33 </td><td> 4.17 </td><td> 0.052 </td></tr></tbody></table> 表1 5月旧Vervetsの右半球の総形態学的測定<img src="/files/ftp_upload/1259/1259fig1.jpg" alt="図1" /> <img src="/files/ftp_upload/1259/1259fig2.jpg" alt="図1.5" /> 図1。脳をブロックするために使用される冠状面の回路図。これは、外部化された成人のベルベット脳の例です。ブロッキング手順の例。ここに縦の線は、通常、それぞれの半球から7冠状ブロックを生成する、1センチメートルで離間している。 <img src="/files/ftp_upload/1259/1259fig3.jpg" alt="図2" /> 図2。定位空間における脳組織のブロックは、各ブロックは、50μmので撮影した約200のセクションが得られます。冠状面でスライスしたときにこのサンプリング方式と皮質を介して1200以上のセクションは、小脳から取られ、追加の400から500になります。

<ol>
	<li>Gallagher, M., Rapp, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+animal+models+to+study+the+effects+of+aging+on+cognition.+Annu+Rev+Psychol+48.">The use of animal models to study the effects of aging on cognition. Annu Rev Psychol 48.</a> , 339-370 (1997).</li><li>Clancy, B., Darlington, R., Finlay, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+developmental+time+across+mammalian+species.">Translating developmental time across mammalian species.</a> Neuroscience. 105, 7-17 (2001).</li><li>Nowakowski, R. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+abeta+vaccine+reduces+central+nervous+system+abeta+levels+in+a+non-human+primate,+the+Caribbean+vervet.">Alzheimer's disease abeta vaccine reduces central nervous system abeta levels in a non-human primate, the Caribbean vervet.</a> Am J Pathol. 165, 283-297 (2004).</li><li>Mash, D. C., Staley, J. K., Doepel, F. M., Young, S. N., Ervin, F. R., Palmour, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+dopamine+transporter+densities+in+alcohol-preferring+vervet+monkeys.">Altered dopamine transporter densities in alcohol-preferring vervet monkeys.</a> Neuroreport. 7, 457-462 (1996).</li><li>Burke, M. W., Palmour, R. M., Ervin, F. 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The authors have nothing to disclose.

セントキッツベルベットモンキー（Chlorocebus aethiops sabeusは ）人間のように皮質と皮質下の脳の発達の類似したパターンと速度と旧世界霊長類です。この種は、不安な動作のような複雑な人間の行動障害、高血圧症8、大脳半球切除術9、パーキンソン病10、Alzhemier病11、およびアルコールの乱用12をモデル化するために使用されています。さらに最...

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<td>Scalpel</td>
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dissecting the non-human primate brain in stereotaxic space

ヒト以外の霊長類の使用は、ヒトの発達と老化の理解のための優秀な翻訳モデルを提供<sup&gt; 1月6日</sup&gt;。さらに、ヒト以外の霊長類の使用は最近、自然にそのようなアルコールの乱用などの複雑な精神疾患をモデル化する機会を与えている<sup&gt; 7</sup&gt;。ここでは、ベルベットモンキーの冠状面で脳をブロックするための手法を記述する（<em&gt; Chlorocebus aethiops sabeus</em&gt;）定位空間にそのまま頭蓋骨インチここで説明する方法はブロックと対象とブロックの間を最小限に抑えるの部分のセクションの間にセクションの標準的な平面を提供します。冠状面での組織のブロックの切削することも関心の領域の線引きを容易にします。 50μmの時にスライスするときにベルベットモンキーの単一の半球は、1200以上のセクションをもたらすので、このメソッドは、管理可能なサイズのブロックを提供します。さらに1センチメートルブロックに脳をブロックすることによって、それはcyroprotectionためにショ糖の浸透を容易にし、ブロックが標準的なクライオスタットにスライスすることができます。

Watch this Scientific Journal Video about Dissecting the Non-human Primate Brain in Stereotaxic Space at JoVE.com

Dissecting the Non-human Primate Brain in Stereotaxic Space

ヒト以外の霊長類は脳の正常な処理の理解のための重要な並進種です。霊長類の脳の解剖学的組織は、ヒトの正常と病理学的状態に重要な洞察を提供することができます。

定位空間における非ヒト霊長類の脳を解剖

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

dissecting-the-non-human-primate-brain-in-stereotaxic-space

Research

JoVE Journal

Biology

10.0K ビュー.  University of Montreal. ヒト以外の霊長類は脳の正常な処理の理解のための重要な並進種です。霊長類の脳の解剖学的組織は、ヒトの正常と病理学的状態に重要な洞察を提供することができます。

ビデオ: Dissecting the Non-human Primate Brain in Stereotaxic Space

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

げっ歯類で存続可能な定位手術

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

生物学

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

解離性とC. elegansの神経筋接合部から録音する

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

脳へのゲートウェイ：霊長目の解剖

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased stereology, the method accepted as state-of-the-art for quantification of biological objects in tissue sections2, generates reliable structural data for biological features in the mammalian brain3. The key components of the approach are unbiased (systematic-random) sampling of anatomically defined structures (reference spaces), combined with quantification of cell numbers and size, fiber and capillary lengths, surface areas, regional volumes and spatial distributions of biological objects within the reference space4. Among the advantages of these stereological approaches over previous methods is the avoidance of all known sources of systematic (non-random) error arising from faulty assumptions and non-verifiable models. This study documents a biological application of computerized stereology to estimate the total neuronal population in the frontal cortex of the vervet monkey brain (Chlorocebus aethiops sabeus), with assistance from two commercially available stereology programs, BioQuant Life Sciences and Stereologer (Figure 1). In addition to contrast and comparison of results from both the BioQuant and Stereologer systems, this study provides a detailed protocol for the Stereologer system.

The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given reference space1.

非ヒト霊長類の脳の公平な立体学を：大切なのを知ること

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased ...

Trajectory Data Analyses for Pedestrian Space-time Activity Study

It is well recognized that human movement in the spatial and temporal dimensions has direct influence on disease transmission1-3. An infectious disease typically spreads via contact between infected and susceptible individuals in their overlapped activity spaces. Therefore, daily mobility-activity information can be used as an indicator to measure exposures to risk factors of infection. However, a major difficulty and thus the reason for paucity of studies of infectious disease transmission at the micro scale arise from the lack of detailed individual mobility data. Previously in transportation and tourism research detailed space-time activity data often relied on the time-space diary technique, which requires subjects to actively record their activities in time and space. This is highly demanding for the participants and collaboration from the participants greatly affects the quality of data4.
Modern technologies such as GPS and mobile communications have made possible the automatic collection of trajectory data. The data collected, however, is not ideal for modeling human space-time activities, limited by the accuracies of existing devices. There is also no readily available tool for efficient processing of the data for human behavior study. We present here a suite of methods and an integrated ArcGIS desktop-based visual interface for the pre-processing and spatiotemporal analyses of trajectory data. We provide examples of how such processing may be used to model human space-time activities, especially with error-rich pedestrian trajectory data, that could be useful in public health studies such as infectious disease transmission modeling.
The procedure presented includes pre-processing, trajectory segmentation, activity space characterization, density estimation and visualization, and a few other exploratory analysis methods. Pre-processing is the cleaning of noisy raw trajectory data. We introduce an interactive visual pre-processing interface as well as an automatic module. Trajectory segmentation5 involves the identification of indoor and outdoor parts from pre-processed space-time tracks. Again, both interactive visual segmentation and automatic segmentation are supported. Segmented space-time tracks are then analyzed to derive characteristics of one's activity space such as activity radius etc. Density estimation and visualization are used to examine large amount of trajectory data to model hot spots and interactions. We demonstrate both density surface mapping6 and density volume rendering7. We also include a couple of other exploratory data analyses (EDA) and visualizations tools, such as Google Earth animation support and connection analysis. The suite of analytical as well as visual methods presented in this paper may be applied to any trajectory data for space-time activity studies.

A suite of spatiotemporal processing methods are presented to analyze human trajectory data, such as that collected using a GPS device, for the purpose of modeling pedestrian space-time activities.

歩行者空間·時間·アクティビティ·研究のための軌道データ解析

It is well recognized that human movement in the  spatial and temporal dimensions has direct influence on disease transmission1-3.  An infectious disease ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

ラットの外側尾静脈から血液をサンプリング

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

Micropuncture of Bowman&#039;s Space in Mice Facilitated by 2 Photon Microscopy

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

2 光子顕微鏡による促進マウス Bowman 腔のそれによって

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

郭清と霊長類の顔の進化における DiceCT 明らかにする別の方法で非表示のデータを逆に

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Situハイブリダイゼーションにおける2色単分子蛍光を用いた 大腸菌 における空間と時間におけるmRNA運動学の探査

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force.
By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

超高速フォースクランプ分光法によるミオシンのメカノ酵素的性質の解剖

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...