著者は、彼の技術サポートのためにIkiel Ptitoに感謝します。私たちは、霊長類の仕事の継続的なサポートのために、フランクエルヴィン、ロバータパーマー、セントキッツ、西インド諸島に位置する行動科学財団研究所のスタッフに感謝しています。

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。それは直後には、ちょうどレンズの下に固定液を注入し、固定液に保存されていることが目を犠牲にすることをお勧めします。 </li><li>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、完全に放血するまで0.1MのPBSでtranscardially灌流。これは、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が続いている。 </li></ol>パート2：眼窩から眼球の除去<ol><li>眼球へのアクセスを容易にするためには、まず脳を除去することをお勧めします。脳が削除されると薄肉の軌道の骨が容易に明らかである。徐々に軌道の壁を崩すために骨Rongeursを使用してください。メスで眼の筋肉を切り取ると、眼球からの結合組織を取り除く。慎重に視神経を切断、これは電子顕微鏡の研究に使用することができます。眼球は今眼窩から解放する必要があります。 </li></ol>パート3：アイカップから網膜を解剖<ol><li>乾燥から網膜を保つためにPBSでシャーレに目を置きます。解剖顕微鏡または表では、ライトスタンドを拡大することが必要ではない解剖に有用ですが、マウント。春のはさみと鋸状縁のレベルで角膜の周囲に密接に強膜を切断することにより角膜を取り出して、ピンセットでレンズを取り外します。 </li><li>強膜から網膜を削除するにはペイントブラシ、ピンセット、そして春のはさみを使用してください。これは、強膜から網膜を分離して、はさみで離れて強膜を切断することによって行われます。一つは、網膜組織を損傷したり涙にしないように少しずつでこのプロセスを実行する必要があります。強膜は容易にそう慎重に視神経の周り強膜を切断残骸視神経から分離されていません。 </li><li>この時点で網膜は、その湾曲した形状を保持し、サンプリングのために展開する必要がありますしています。網膜を平坦化する前に、ゼリーの一貫性を持っている硝子は、一括して削除することができます。スライドに網膜を平坦にする、手術用メスの刃を持つ複数の放射状のカットを行います。残留硝子のユーモアは、今普通のフィルター紙と絵筆を用いて除去することができます。それは硝子体液を除去するときに穏やかであることが不可欠であるので、網膜神経節細胞層は、この時点で公開されます。視神経がまだ視神経乳頭に接続されている場合は、手術用メスの刃と春のはさみを使って網膜をリッピングすることなく視神経を削除します。これは、現在のフラットマウントの網膜（図1）であり、中心窩は視神経乳頭からの時間/腹側方向に暗いパッチとして明らかなはずである。 </li></ol>第4部：サンプリング<ol><li>網膜をサンプリングするためのオプションがいくつかあります。ここでは、flatmount準備とisodentricサンプリングを説明します。両方の手順については離れてスライドから視神経繊維層と網膜をflatmount。意図はflatmount準備の網膜神経節細胞層を調べることであれば、それはどちら色素上皮を除去または漂白する必要があります。色素上皮は、この層の一部を削除するために軽くブラシを使用して削除するには、これは、感光体層を破壊する傾向があります。漂白は、1時間過マンガン酸カリウム溶液の網膜（0.25％）を浸漬し、蒸留水で洗浄し、5分間シュウ酸（5％）でクリア伴います。2 </li><li>意図が網膜全体に各レイヤのセルの分布と形態を検討するならば、我々は、網膜の等角サンプリングを示唆している。網膜は、スライド上flatmountedとPBSで湿った保つ必要があります。アイソメトリックサンプリングの場合は、鼻の頭、上下方向（図1）における視神経乳頭から等距離組織の小片をカット。それはこの準備の漂白剤色素上皮をする必要はありません。これらの作品は現在、クライオスタット、ビブラトーム、または研究の質問に応じて、ウルトラミクロトームによる冠状面で区分けすることができます。クリオスタット切片の場合、作品は一晩30％ショ糖で凍結保護されるべきであり、ドライアイス上で封入剤のコンテキストで凍結。また、サンプルは寒天に埋め込まれ、ビブラトームでスライスできます。クライオスタットとビブラトーム準​​備が確実に4メートルほどの薄いセクションが得られます薄いセクション（電子顕微鏡の準備用など）が必要な場合は、それは、ウルトラミクロトーム用試料を調製する必要がある。これを行うには、セクションでは、fumehoodの下で1時間オスミウム四酸化で後固定してください。これは、段階的エタノール系列で脱水（50、70、95、95、100、100％）と100％のプロピレンオキサイドが続いている。組織その後、エポン（EMBED - 812埋め込みキット）に組み込まれています。 </li></ol>パート5：代表的な結果： 当研究室では、我々は日常的にcryosectioned網膜（図2）で免疫組織化学を行う。この場合、我々は、霊長類の網膜におけるカンナビノイド受容体（CB1）の等密度に興味を持っています。我々はまた、霊長類の視覚システム上の出生前のエタノール曝露の影響を調べる。この目的のために、我々は、中心窩でと周辺網膜における細胞密度と層の厚さに興味を持っています。これを達成するために、我々は、ウルトラミクロトームで700nmの時、エポンでスライスを網膜の断片を埋め込む、と1％トルイジンブルー（図3）で染色。 <img src="/files/ftp_upload/1261/fig1.jpg" alt="図1" /> 図1 Flatmount網膜。ラジアルカットは網膜を平坦にするために使用されます。 <img src="/files/ftp_upload/1261/fig2.jpg" alt="図2" /> 図2免疫染色。末梢網膜の凍結切片は、網膜神経節細胞が大きく内側と外側核の層のいくつかのラベルで標識されているCB1を免疫染色。このセクションの厚さはスライド上の14メートルとステンドグラスです。 RG - 網膜神経層と、IP - 内網状層、IN - 内顆粒層、OP - 外網状層、ON - 外核層。 <img src="/files/ftp_upload/1261/fig3.jpg" alt="図3" /> エポンに埋め込 ​​まれ、700nmのでultramicrotromeにスライスされた窩を通じて、 図3中心窩。セクション。光受容体層（PR）は、セクションを位置合わせするために使用された。光受容体の全範囲が冠状面で適切な角度を示す識別できることに注意してください。密度の測定は300メートル、500メートル、そしてBioquantイメージングシステムを用いた中心窩ピットの中心から800mで採取した。 RG - 網膜神経層と、IP - 内網状層、IN - 内顆粒層、OP - 外網状層、ON - 外顆粒層、スケールバー= 50メートル

<ol>
	<li>Hendrickson, A., Kupfer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histogenesis+of+the+fovea+in+the+macaque+monkey.">The histogenesis of the fovea in the macaque monkey.</a> Invest Ophthalmol Vis Sci. 15, 746-756 (1976).</li><li>Herbin, M., Boire, D., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+and+distribution+of+retinal+ganglion+cells+in+the+St.+Kitts+green+monkey+(Cercopithecus+aethiops+sabeus).">Size and distribution of retinal ganglion cells in the St. Kitts green monkey (Cercopithecus aethiops sabeus).</a> J Comp Neurol. 383, 459-472 (1997).</li><li>Stone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Wholemount Handbook. , (1981).</li><li>Herbin, M., Boire, D., Theoret, H., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transneuronal+degeneration+of+retinal+ganglion+cells+in+early+hemispherectomized+monkeys.">Transneuronal degeneration of retinal ganglion cells in early hemispherectomized monkeys.</a> Neuroreport. 10, 1447-1452 (1999).</li><li>Krebs, W., Krebs, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primate Retina and Choroid Atlas of Fine Structure in Man and Monkey. , (1991).</li><li>Pow, D., Sullivan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+kinesis,+neurite+sprouting+and+abnormal+axonal+projections+of+cone+photoreceptors+in+the+aged+and+AMD-afflicted+human+retina.">Nuclear kinesis, neurite sprouting and abnormal axonal projections of cone photoreceptors in the aged and AMD-afflicted human retina.</a> Exp Eye Res. 84, 850-857 (2007).</li></ol>

wholemountとして網膜の調製は、神経節細胞層や網膜血管3の内皮細胞のいずれかの地形と空間分布の解析が可能になります。霊長類の網膜の周辺部における細胞密度の定量化は容易に達成される。しかし、perifovealと中心窩の地域では、神経節細胞層で複数の層のスタッキングでは、定量化を妨げ。この潜在的なバイアスを回避するために、中心窩とperifoveal領域は、冠状面2,4に?...

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the gateway to the brain: dissecting the primate eye

ヒトの視覚系は、世界への入り口と考えられ、感覚、知覚や認知過程の過多で主要な役割を果たしている。それは視力の質、生活の質に結びついていることは驚くことではありません。視覚障害の原因を取り巻く広範な臨床および基礎研究にもかかわらず、このような網膜色素変性症や黄斑変性症などの視覚障害、、多くの形態では、効果的な治療法を欠いている。ヒト以外の霊長類はヒトと眼の発生の最も近い一般的な機能を備えています。だけでなく、彼らは同様の血管解剖を持っていますが、他の哺乳動物の中で、霊長類は、高視力に特化した耳側網膜に領域を有する固有の特性、中心窩を持っているか<sup&gt; 1</sup&gt;。ここでは、網膜の組織学、免疫組織化学、レーザーキャプチャーマイクロダイセクションだけでなく、光や電子顕微鏡のための組織を提供するために、霊長類の網膜を切開するための一般的な手法を説明します。翻訳モデルとしての非ヒト霊長類の長期使用と、私たちの希望は、網膜の理解の向上が影響を受ける個人の生活の質に視覚障害のマイナスの影響を減衰または反転に向けた効果的なアプローチへの洞察を提供するです。

Watch this Scientific Journal Video about The Gateway to the Brain: Dissecting the Primate Eye at JoVE.com

The Gateway to the Brain: Dissecting the Primate Eye

ヒト以外の霊長類は、開発と老化の理解のための重要な並進種です。霊長類の網膜の解剖学的組織は、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あります。

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 ...

the-gateway-to-the-brain-dissecting-the-primate-eye

Research

JoVE Journal

Biology

14.1K ビュー.  University of Montreal. ヒト以外の霊長類は、開発と老化の理解のための重要な並進種です。霊長類の網膜の解剖学的組織は、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あります。

ビデオ: The Gateway to the Brain: Dissecting the Primate Eye

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 ...

生物学

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

レンズゼブラフィッシュでの移植とアイ変異体の解析への応用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Dissecting the Non-human Primate Brain in Stereotaxic Space

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In addition, the use of non-human primates has recently afforded the opportunity to naturally model complex psychiatric disorders such as alcohol abuse7. Here we describe a technique for blocking the brain in the coronal plane of the vervet monkey (Chlorocebus aethiops sabeus) in the intact skull in stereotaxic space. The method described here provides a standard plane of section between blocks and subjects and minimizes partial sections between blocks. Sectioning a block of tissue in the coronal plane also facilitates the delineation of an area of interest. This method provides manageable sized blocks since a single hemisphere of the vervet monkey yields more than 1200 sections when slicing at 50&mu;m. Furthermore by blocking the brain into 1cm blocks, it facilitates penetration of sucrose for cyroprotection and allows the block to be sliced on a standard cryostat.

The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans.

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 ...

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 ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

のアイ成虫原基の可視化を支援するために新しいRFPレポーターショウジョウバエ</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

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, ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

裏打ちクリア: 組織の脂肪組織の三次元イメージング法をクリア

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

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 ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

マウスから脳ペリサイトを分離する高出力法

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

蚊のエレクトロアンテノグラフィーのステップバイステップガイド

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...