我々は、BAC GLT1たeGFPマウスとGLT1抗体を提供するための博士ジェフリー·ロススタインに感謝したいと思います。貴重なコア機能を提供するため、脳科学研究のためのタフツセンター（PI、ロブ·ジャクソンNIH P30 NS047243）;新教員採用助成金（NIH P30 5P30NS069254-02 ;タフツ神経科学部門のPI、フィル·ヘイドン）。

1。マイクロ流体培養チャンバー（MCP）の組立<ol><li> MCP（ 図1）はセル4の異なる種類の区画文化のために設計されたオープン室である。これは、典型的には、中央のチャネル（直径3μm）を介して接続されている2つの区画を持っています。ガラス底皿とMCPの組み立ては、文化やその後の画像解析を準備するために必要です。 </li><li>まず、オルニチン（Sigma-Aldrich社、1 mg / ml）を持つコート無菌のガラス底皿は四ホウ酸ナトリウム（Sigma-Aldrich社、10mMのpH8.5）に溶解し、37℃で一晩インキュベートした。 </li><li>次の日に、コーティングされたガラス底皿をddH 2 Oで3回洗浄し、滅菌ヒュームフードの下で乾燥させた。ガラス底皿はその後さらにラミニン（シグマアルドリッチは、1 mg / ml）をコーティングし、1時間UV光下で乾燥した。コー​​ティングされた料理は、使用する準備ができているか、使用するまで-20℃で保存することができます。これらのコーティング共培養におけるめっきニューロンおよびアストロサイトのために必要な再。 </li><li>文化プラットフォームを組み立てるために、マイクロ流体文化プラットフォームはタイトなシールを形成するために、その下側の中央の接続チャネルでコーティングされたガラス底皿の上に置かれた。定期的なニューロンやアストロサイトの培養培地を組み立てMCPに両側（ 図1）（細胞播種領域から）を添加し、37℃で2-4時間インキュベートし、MCPとガラスの間に漏れがないことを確認したボトムディッシュ。私たちには漏れがないことを確認するために細胞をプレーティングする前に培地をテストしている。ガラスボトムディッシュにMCPの組み立てが新たに使用する前に準備する必要があります。 </li></ol> 2。組立てMCPの神経軸索の神経文化と誘導の準備<ol><li>大脳皮質の神経細胞培養物は、新鮮胚の14から16日齢マウスの脳から調製した。神経細胞培養培地をNeurobasal培地、2％B27 neurobasaから構成されている100xのグルタマックスの1％、1％ペニシリン - streptomysinを追加することで、リットルサプリメント、2mMグルタミン。マウスの脳の解剖の後、髄膜を解剖顕微鏡下で皮質から削除されました。組織は、トリプシンにさらされる表面積を増やすために、小さな組織ブロックを作るためにかみそりの刃でミン​​チにしました。組織ブロックはファイアーポリッシュパスツールピペットで粉砕して穏やかに解離した後、37℃の水浴中で10分間（Sigma-Aldrich社、0.05％トリプシン）トリプシン処理し、及びました。解離された細胞は、明確な神経細胞懸濁液を収集するために70μmのストレーナーを通して濾過した。 </li><li>そのニューロンは細胞保持領域（ 図1）に接続できるような神経細胞（2-3×10 6個 / ml、150μl）を組み立てたMCPの右側のセルメッキ領域（ 図1）上にプレーティングした。作りたてのニューロンは、レギュレーションに5％ウシ胎児血清（Hyclone）を補充しているニューロンメッキ培地で播種したRニューロン培養培地。細胞保持領域にアタッチのみニューロンが中央のチャネルを介して組み立てられたMCPの反対側に軸索を成長することができるので、それだけでは十分ではニューロンが接続されるように細胞播種面積のニューロンのプレートに高密度に重要である細胞保持領域。神経軸索の高密度の代表画像を図2Aに示されています。 </li><li>翌日、ニューロンメッキ媒体はレギュラーニューロン培養培地と交換した。変化媒体は慎重に細胞保持​​エリアと中央の接続チャネル内の気泡を避けるために、チャンバーの細胞播種エリアからではなく、組み立てたMCPの細胞保持領域から培地を吸引することによって達成された。気泡が厳しく軸索伸長とMCPにおいて中心的なチャネルに次のエントリを阻害するであろう。グリア細胞由来神経栄養因子（GDNF、10〜20 ng / mlの）も、他の側（左側）に追加されました組み立てられたMCP（ 図2A）の中央チャンネルを通じて神経側と十字架から軸索伸長5の誘導を容易にするために同じ日にチャンバーの。 GDNFをせずに自発的な神経突起伸長のかなりの量はまた組み立てられたMCPの中央チャンネルを通じて神経側とクロスから観察される。中央のチャンネルに入力した軸索は、多くの場合2〜3日、めっき後に観察される。軸索は中央のチャンネルに入ると、彼らは通常、1日以内にもう一方の側を入力します。軸索は、反対側を入力した後、少なくとも1週間は正常に変更されていない。 </li></ol> 3。コンパートメント化された共培養系を確立するために、MCPに培養アストロサイトの追加プライマリアストロサイトの培養は、P1-3マウス子犬脳から調製した。脳解離手順は、上記の神経細胞の単離操作に似ています。アストロサイトは、まず事前た10cmの皿に播種したオルニチン（シグマアルドリッチは、1 mg / ml）でコーティングした。星状膠細胞培養培地（DMEM、10％ウシ胎児血清、1％ペニシリン - streptomysin）破片を除去するために次の2日間、毎日変更されました。その後、培地を3日ごとに変更されました。 <ol><li>アストロサイトは、90％コンフルエントになると7日後に単分子膜を形成します。コンフルエントアストロサイトをトリプシン処理し、軸索が入ろうとしている、または単にの左側に入力したときに再懸濁した星状細胞（1×10 6個 / ml）150μlを組み立てたMCPの左側のセルメッキ面積に再播種したMCPの組み立て。アストロサイトは、通常、神経細胞が播種した4-5日後に播種した。 GDNFは最初MCPの左側にアストロサイトの再メッキ前に削除されました。 GFAPの免疫染色（ 図1）で示されるように、再メッキアストロサイトは、細胞保持領域に付着させた。チャンバーの両側の間の媒質の唯一の最小流量（蛍光dによって決定はい）は、以前に4,6に示すように 、MCPに発見された。 </li><li>アストロサイトは、90％コンフルエントになると7日後に単分子膜を形成します。コンフルエントアストロサイトをトリプシン処理し、軸索が左に先ほど入力を入力するか、または持ってしようとしているときに再懸濁した星状細胞（1×10 6個 / ml）150μlを組み立てたMCPの左側のセルメッキ面積に再播種した組み立てられたMCPの側。アストロサイトは、通常、神経細胞が播種した4-5日後に播種した。 GDNFは最初MCPの左側にアストロサイトの再メッキ前に削除されました。 GFAPの免疫染色（ 図1）で示されるように、再メッキアストロサイトは、細胞保持領域に付着させた。以前に4,6に示すようにチャンバーの両側の間の媒質の唯一の最小流量は、（蛍光色素によって決定される）は、MCPで発見された。 </li><li>アストロサイトの再めっき後、組み立てられたMCPの左側に入った軸索は、直接astrと対話可溶性因子の直接の軸索の接触またはreleaseによってocytes。アストログリア細胞膜のグルタミン酸トランスポーターと神経GLT1βIII-チューブリンの免疫染色すると、 図2D-Fに示すように、軸索とアストロサイトとの間の相互作用を可視化するために行われた。 </li></ol>

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特別な利害関係は宣言されません。

MCPベースのニューロンとアストロサイトの共培養系は、軸索のみが中央のチャンネルを通過できるようにし、星状膠細胞と相互作用することにより、星状膠細胞シグナル伝達経路の詳細な神経細胞の解剖を可能にします。この共培養系は、便利な、従来のニューロンとアストロサイトの培養の手順で設定することができます。我々はまた、アストロサイトにおける軸索依存GLT1プロモーター?...

<table border="1"><tbody><tr><td>ウシ胎仔血清</td><td> Hyclone社</td><td> SH30070.03 </td><td>ニューロンcutlure媒体のニューロンをメッキする</td></tr><tr><td>ウシ胎仔血清</td><td>シグマアルドリッチ</td><td> F4135 </td><td>アストロ培地の</td></tr><tr><td>グリア細胞由来神経因子</td><td> R＆Dシステム</td><td> 212-GD </td><td>チャンバーのニューロン側に10から20 ng / mLを適用</td></tr><tr><td>ダルベッコ改変イーグル培地高グルコース</td><td>シグマアルドリッチ</td><td> 11995 </td><td></td></tr><tr><td> 70ミリメートルのセルストレーナー</td><td> BDファルコン</td><td> 352350 </td><td></td></tr><tr><td>無菌のガラスボトムディッシュ</td><td>マテック株式会社</td><td></td><td></td></tr><tr><td>マイクロ流体文化プラットフォーム</td><td>ゾナマイクロフルイディクスLLC </td><td> SND150 </td><td></td></tr><tr><td>培養プレートの6ウェル</TD&gt; <td> Cellstar </td><td> 657 160 </td><td></td></tr><tr><td></td><td></td><td></td><td> ニューロン培養培地 <ul><li> Neurobasal培地</li><li> 2パーセントB27 Neurobasalサプリメント</li><li> 100xのグルタマックス1％を追加することにより、2 mMのグルタミン酸</li><li> 1％ペニシリン - streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> めっきセルのためのニューロン培養培地 <ul><li> Neurobasal培地</li><li> 2パーセントB27 Neurobasalサプリメント</li><li> 100xのグルタマックス1％を追加することにより、2 mMのグルタミン酸</li><li> 1％ペニシリン - streptomysin </li><li> 5％ウシ胎児血清SH30070.03 </li></ul></td></tr><tr><td></td><td></td><td></td><td> アストロ培地 <ul><li>ダルベッコ改変EAGLE高グルコース培地</li><li> 10％ウシ胎児血清F4135 </li><li> 1％ペニシリン - streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> 表1マイクロ流体文化プラットフォームベースの神経軸索とグリア共培養系で使用される材料。 </td></tr></tbody></table>

imaging analysis of neuron to glia interaction in microfluidic culture platform (mcp)-based neuronal axon and glia co-culture system

グリア相互作用への適切なニューロンは、中枢神経系（CNS）の生理機能に重要である。この双方向通信は高度にニューロンとグリア1,2の間の特定のシグナル伝達経路によって媒介される。これらのシグナル伝達経路の同定および特徴は、グリア相互作用の形中枢神経生理学にどのニューロンの理解に不可欠である。以前は、ニューロンとグリア細胞混合培養が広くニューロンとグリア細胞間のシグナル伝達経路をテストおよび特性評価に利用されてきた。我々 は 、in vivo のツールで 、これらの製剤と他から学んだことは、しかし、ニューロンとグリア細胞間の相互シグナリングがしばしばニューロン（ すなわち 、軸索、樹状突起、またはソーマ）3内の特定の区画で発生したことを示唆している。これは、重要な神経区画の分離を可能にする新しい培養システムを開発することができ、具体的にはグリア細胞と神経の間の相互作用を調べNAL軸索/樹状突起。加えて、従来の混合培養系は、可溶性因子とニューロンとグリア細胞間の直接メンブレン接点信号を区別することができるではありません。さらに、従来の共培養系におけるニューロンとグリア細胞の大量は、単一の軸索とグリア細胞間の相互作用を観察するために必要な決議を欠いている。 そこで本研究では、マイクロ流体文化プラットフォーム（MCP）を用いた新規軸索とグリア共培養系を記述します。この共培養系では、神経細胞とグリア細胞は、複数の中央のチャネルで接続されている2つの別々の室で培養される。このマイクロ流体文化のプラットフォームでは、唯一の神経プロセス（特に軸索）は、中央のチャネルを介してグリア側を入力することができます。強力な蛍光タンパク質の標識との組み合わせでは、このシステムはそのような、軸索/樹状細胞およびグリア相互作用の間のシグナル伝達経路を直接検査することができます神経端末におけるグリア、グリア媒介受容体の輸送、およびグリア媒介軸索成長のs軸索を介した転写調節。チャンバーの狭い直径も大きく軸索/樹状突起とグリアの表面との間の直接の膜タンパク質相互作用のプロービングを容易にして、グリアチャンバー内にニューロンに富んだ媒体の流れを禁止しています。

アストロサイトにおける軸索誘導のGLT1プロモーター活性化のタイムラプスイメージング解析コンパートメントニューロンとアストロサイトの共培養システムは、特に唯一の神経突起、軸索は、選択的にアストロサイトと対話することができます。組み立てMCPにおける軸索と星状膠細胞（または他のグリア細胞）の共培養の成功確立に続いて、軸索·グリア相互作用の異なる...

Watch this Scientific Journal Video about Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System at JoVE.com

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

この研究では、神経細胞の軸索と小説（ASTRO）グリア共培養プラットフォームをセットアップする手順について説明します。この共培養系では、単一の軸索（と単一神経膠細胞）との間の直接的な相互作用の操作は、グリアシグナリング相互ニューロンのメカニズム解析を可能にすることが可能となる。

マイクロ流体文化·プラットフォーム（MCP）ベースの神経軸索とグリア共培養系におけるグリア相互作用へのニューロンのイメージング解析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

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Neuroscience

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

14.9K ビュー.  Tufts University. この研究では、神経細胞の軸索と小説（ASTRO）グリア共培養プラットフォームをセットアップする手順について説明します。この共培養系では、単一の軸索（と単一神経膠細胞）との間の直接的な相互作用の操作は、グリアシグナリング相互ニューロンのメカニズム解析を可能にすることが可能となる。

ビデオ: Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

初代ラット皮質ニューロンとグリアのBilaminarの共培養

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

神経科学

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

マイクロ流体共培養装置の使用歯胚神経支配の開発の分析

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

間接ニューロン、星状細胞共培養アッセイ：<em&gt;インビトロ</em&gt;ニューロングリアの相互作用の詳細な調査のためのセットアップ

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

成人ラット尿膀胱からの一次ニューロンとグリアの分離と培養

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

脳回路の非侵襲的切断に対する標的ニューロン損傷

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

複数の中枢神経系領域からのマウスグリアの解剖と分離

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

虚血におけるニューロングリア相互作用の役割を研究するための細胞培養モデル

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

インビボ マウス劣オリーブにおけるカルシウムイメージング

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

カルシウムイメージング技術によるニューロン-グリア回路の変化の可視化

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

ヒト人工多能性幹細胞由来ミクログリアの免疫適格マウス脳への非侵襲的経鼻経路による移植

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...