我々は優雅にスクリプト用ドクタージョンヘイズに感謝;博士。エリック·ブラッドリーと共焦点顕微鏡の支援のためのクリストファー·デル·ネグロ、ドリュー·ヒューズ、プロジェクトを開発し、予備的なデータを提供する際に自分の仕事のためラウラOdorizzi、アレックスGarafalo、レベッカラウデン、とリズ·マクマレイ、統計分析に役立つ提案のための博士グレッグスミス。この作品は、MSSにNIHの助成金（NINDS IR15N5067566-01）とウィリアム·アンド·メアリー大学のハワードヒューズ医学研究所の科学教育助成金によって支えられている。

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すべての脊椎動物全体で保存されており、その十分に特徴付けられた細胞の種類によって、網膜は細胞型仕様と分化を支配する分子細胞過程を研究するための有用なモデルを提供します。初代培養細胞は、遺伝子発現、蛋白質のダイナミクス、およびカルシウム、単一細胞の解像度レベルでの電気的活動を含むプロセスの広い範囲を調査するための強力な方法を与える。ここでは、 アフ?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td></td></tr><tr><td></td><td></td><td></td><td> 解剖と培養するための </td></tr><tr><td> BDファルコンイージーグリップ組織培養皿、35ミリメートル</td><td>フィッシャー</td><td> 08-772A </td><td></td></tr><tr><td>使い捨てのポリスチレンペトリ皿、60×15ミリメートル</td><td>フィッシャー</td><td> 0875713A </td><td></td></tr><tr><td> 35ミリメートルNunclon表面ペトリ皿（Airvent付き） </td><td>フィッシャー</td><td> 12-565-91 </td><td></td></tr><tr><td>デュモンファイン鉗子（Dumostar＃55） </td><td>フィッシャー</td><td> NC9341917 </td><td></td></tr><tr><td> Cellatticeマイクロ支配プラスチックカバースリップ、25ミリメートル</td><td>フィッシャー</td><td> 50-313-17 </td><td></td></tr><tr><td>エチル-m-アミノ安息香酸メタンスルホン酸塩（MS-222） </td><td> MPバイオメディカル、LLC </td><td> 103106 </td><td></td></tr><tr><td>硫酸ゲンタマイシン</td><td>エンツォ生命科学</td><td> 380から003-G025 </td><td></td></tr><tr><td>ギブコトリプシン（1:250）パウダー</td><td>ライフテクノロジーズ</td><td> 27250-018 </td><td></td></tr><tr><td> クロストリジウム·ヒストリチカムからのコラゲナーゼB </td><td>ロッシュ</td><td> 11088831001 </td><td></td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td>ギブコ</td><td> 15140-122 </td><td></td></tr><tr><td>ナイルブルー硫酸（オプション） </td><td>ファルツ＆バウアー</td><td> N05550 </td><td></td></tr><tr><td> 500mlの真空フィルター/ストレージボトルシステム、孔径0.22μm33.2センチメートル2 CN膜</td><td>コー​​ニング</td><td> 430758 </td><td></td></tr><tr><td></td><td> &lt;/ TD&gt; <td></td><td> カルシウムイメージングのための </td></tr><tr><td>のFluo-4は、DMSOで1mM溶液、永久細胞AM </td><td>ライフテクノロジーズ</td><td> F-14217 </td><td></td></tr><tr><td>水中でのプルロニックF-127の10％溶液</td><td>ライフテクノロジーズ</td><td> P-6866 </td><td></td></tr><tr><td> LSM 510共焦点顕微鏡システム</td><td>ツァイス</td><td>モデル廃止</td><td></td></tr><tr><td>ブロッキング試薬</td><td>ロッシュ</td><td> 11096176001 </td><td></td></tr><tr><td></td><td></td><td></td><td> 蛍光in situハイブリダイゼーション （FISH）を用 </td></tr><tr><td>抗ジゴキシゲニン-POD、Fab断片</td><td>ロッシュ</td><td> 11207733910 </td><td></td></tr><tr><td>抗DNP-HRP抗体</td><td>パーキンエルマー</td><td> NEL747A </td><td></td></tr><tr><td> Cy3でNHSエステル</td><td> GEヘルスケア</td><td> PA13101 </td><td></td></tr><tr><td> NHS-フルオレセイン</td><td>サーモサイエンティフィック</td><td> 46409 </td><td></td></tr><tr><td>ホルムアミド、脱イオン</td><td> Amresco </td><td> 0606-950ML </td><td></td></tr><tr><td>トルラRNA、IX型</td><td>シグマアルドリッチ</td><td> R3629 </td><td></td></tr><tr><td>ブタ腸粘膜からヘパリンナトリウム塩、 </td><td>シグマアルドリッチ</td><td> H3393-250KU </td><td></td></tr><tr><td> CHAPS </td><td>シグマアルドリッチ</td><td> C3023 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表2に特異的な試薬や機器。 </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> ソリューション </td><td> リファレンス </td><td> 中身 </td></tr><tr><td>細胞培養培地</td><td>チャンとスピッツァー、2009 54 </td><td> 116 mMのNaCl 0.67のKCl 2mMのCaCl 2 1.31 mMのMgSO 4を 4.6 mMトリス 1％（v：v）のペニシリン/ストレプトマイシン HClで7.8にpHを調整する。 0.22μmのCNのフィルターを通過することによって殺菌をフィルタリングします。 4℃で保存する</td></tr><tr><td>カルシウム·マグネシウム培地（CMF） </td><td>グーら 、1994 42;。Guとスピッツァー、1995年55 </td><td> 116 mMのNaCl 0.67のKCl 4.6 mMトリス 0.4 mMのEDTA 1％（v：v）のペニシリン/ストレプトマイシン HClで7.8にpHを調整する。 0.22μmのCNのフィルターを通過することによって殺菌をフィルタリングします。 4℃で保存する</td></tr><tr><td>マレイン酸バッファ（MAB） </td><td> Sive ら 、2000年53 </td><td> 100mMのマレイン酸 150mMのNaCl（pH7.5）で。 </td></tr><tr><td> 10X Marcの修正リンガー（MMR） </td><td> Sive ら 、2000年53 </td><td> 100mMのNaCl のKCl 1mMのMgSO 4を 2mMのCaCl 2 5mMのHEPES pHはNaOHで7.4に調整 0.1X MMRは、50 mg / mlの硫酸ゲンタマイシン及びpHをNaOHで7.4に調整されているが含まれています。 </td></tr><tr><td> 10X MEMFAソリューション、 </td><td> Sive ら 、2000年53 </td><td>の0.1M MOPS緩衝液（pH7.4） 2mMのEGTA 1mMのMgSO 4を 3.7％ホルムアルデヒド 10X溶液は、ホルムアルデヒドことなく、4℃で保存することができますホルムアルデヒドは（標準37％ストックの1/10量）新鮮追加されます。 </td></tr><tr><td> in situハイブリダイゼーション緩衝液中に </td><td> SIV電子ら 、2000年53 </td><td> 50％ホルムアミド 5X SSC 1 mg / mlのトルラRNA 100 mg / mlのヘパリン 1Xデンハルト溶液 0.1％のTween20 0.1％CHAPS 10mMのEDTA。 </td></tr></tbody></table>ペニシリンとストレプトマイシンが我々の文化メディアで使用されている間ソリューションを提供しています。*ゲンタマイシン、抗生物質は、私たちのMMRのソリューションで使用されています。 </td></tr></tbody></table>

dissection, culture, and analysis of xenopus laevis embryonic retinal tissue

神経板の前方領域は脊椎動物の網膜を生じさせることによって、プロセスは、臨床と基礎研究の両方の主要な焦点であり続けている。網膜疾患の理解と治療のための明白な医学的関連に加えて、脊椎動物の網膜の開発は、神経細胞の種類の判定や分化1-16を理解する上で重要とエレガントなモデルシステムとして機能し続けています。神経網膜には、ステレオタイプのレイヤーに配置された6つの個別の細胞種（神経節、水平、アマクリン視細胞、双極細胞、ミュラーグリア細胞）が、大部分はすべての脊椎動物12,14-18間で保存されているパターンで構成されています。 無傷の胚で網膜を勉強しながら、明らかにこの複雑な器官が層状構造に前脳の突起から発達する方法、あちこち恩恵多くの質問があります理解するために必要とされるmは推定網膜細胞の初代培養細胞を用いた7,19-23アプローチを採用する。たとえば、さまざまな段階で除去され、解離組織から細胞を分析すると、1つは、異なる発達段階で個々のセルの仕様の状態を認識することができ、すなわち、隣接組織8,19-22との相互作用の非存在下における細胞の運命,24-33。初代培養細胞はまた、治験責任医師は、特定の試薬 ​​と文化を扱い、単一細胞レベル5,8,21,24,27-30,33-39に結果を分析することができます。 アフリカツメガエルの研究のための古典的なモデル系初期の神経発達19,27,29,31-32,40-42は 、網膜の初代細胞培養10,38,43-45に特に適したシステムとしての役割を果たします。 推定網膜組織はすぐに神経誘導25,38,43に続いて、開発の初期段階からのアクセスが可能です。また、与えられた目卵黄の供給が含まれている胚の各セルに、網膜細胞は、このようにインキュベーションや他の血清ベースの製品10,24,44-45の交絡影響を除去、緩衝塩溶液からなる非常にシンプルに定義された培地で培養することができる。 しかし、周囲の組織とその後の処理から網膜組織の分離は困難である。ここで、我々は、順番に、単一細胞の解像度でカルシウム活性と遺伝子発現について分析される初代培養細胞を調製するために使用されるアフリカツメガエルの網膜の細胞の解離と解離するための方法を提示する。本稿で紹介トピックが自発カルシウムトランジェントの分析ですが、技術が研究課題とアプローチ（ 図1）の広い配列に広く適用可能である。

首尾よく解剖オプティック胞（ステージ25）、網膜（ステージ35）の例は2Eおよび2J図に示されている。このプロトコルは、開発のさまざまな段階で使用することができますが、それはさらなる実験のために正確さを保証する唯一の網膜組織を得ることが重要です。慎重にすべての段階で表皮を取り除き、あなたの鉗子は網膜組織を穿刺しないことを確認してください。ステージ35?...

Watch this Scientific Journal Video about Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue at JoVE.com

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

アフリカツメガエルは、細胞運命の仕様および初代培養細胞内の個々の網膜細胞の生理機能を研究するための理想的なモデルシステムを提供します。ここでは、網膜組織を解剖し、カルシウム活性のために画像化し、in situハイブリダイゼーションにより分析される初代培養細胞を生成するための手法を提示する。

の解離、文化、および分析<em&gt;アフリカツメガエル</em&gt;胚性網膜組織

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

dissection-culture-analysis-xenopus-laevis-embryonic-retinal

Research

JoVE Journal

Neuroscience

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

13.8K ビュー.  College of William and Mary. アフリカツメガエルは、細胞運命の仕様および初代培養細胞内の個々の網膜細胞の生理機能を研究するための理想的なモデルシステムを提供します。ここでは、網膜組織を解剖し、カルシウム活性のために画像化し、in situハイブリダイゼーションにより分析される初代培養細胞を生成するための手法を提示する。

ビデオ: Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

胚の脊髄から交連ニューロンの解剖と文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

神経科学

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofectionとマウス胎児海馬および皮質ニューロンの初代培養

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

網膜色素上皮の全マウントするためのマウスの眼の解剖

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

マウス胚の運動ニューロンのレクチンに基づく分離と文化

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

ラット胚神経細胞の分離と文化：クイックプロトコル

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

からの神経外植文化<em&gt;アフリカツメガエル</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

胚前脳からの内皮細胞の単離および培養

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

テスト視機能のための簡単​​な行動アッセイ<em&gt;アフリカツメガエル</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

の嗅球にカルシウム変化をモニタリングを通じて温度によって誘発される神経活動を記録<em&gt;アフリカツメガエル</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

準備やアフリカツメガエルの視蓋ニューロンの全体セル パッチ ・ クランプ記録のためのプロトコル

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...