This research was support by an Aims2Cure, UK and a UCL Impact Award Ph.D. studentship to JMP and an MRC Capacity Building Ph.D. studentship in Dementia to JMP.

本明細書に記載の全ての実験は、1986年の英国動物（科学的手続）法に基づいて実施しました。 楽器、文化メディア、および料理の作製<ol><li> 2つのステンレス鋼実験室解剖ハサミと2つのステンレス鋼実験用鉗子を準備します。紫外線下で培養フードO / Nですべての機器と場所をオートクレーブ滅菌用（UV）光。 </li><li>最小必須培地（MEM）培地（10％ウシ胎児血清[FBS]、20mMの塩化カリウム、25mMのNaHCO 3を、30mMのD-グルコース、2mMのL-グルタミン、100 U / mlペニシリン、100 500mlのを作ります/ mlのストレプトマイシンおよび6 / mlのアンピシリン）とフィルター滅菌します。ヶ月間4℃で保存してください。 </li><li> 14ミリメートル、数1の厚さのカバースリップを含む24ウェル培養プレートを準備します。先に説明したように3 100 mg / Lのポリ-D-リジン（PDL）でカバースリップおよびコーティングをオートクレーブ。 UV光はO / Nを殺菌します。 </li></ol> CGC培養液の調製<ol><li>処理されたオートクレーブ処理、UV光の「溶液A」（100ミリリットル）、グルコース250mgのを含有する無菌のdH 2 O、ウシ血清アルブミン[BSA]、リン酸緩衝生理食塩水[PBS]（のCa 2+の955 mgの300 mgを準備し、 MG）+自由、および3.8％の1ミリリットルのMgSO 4·7H 2 O </li><li> 5 mg / mlのトリプシン溶液Aの9ミリリットル中に希釈し、1 mlを含む準備「溶液B」（10ミリリットル） </li><li> 「溶液C」1000個のDNアーゼ、大豆トリプシン阻害剤0.5mgの、溶液Aで10mlに希釈した3.8％のMgSO 4·7H 2 Oの100μLを含む（10ml）に準備</li><li>溶液C 3.2 mlを含有する「溶液D &#39;（20ml）を溶液Aで20mlまで希釈し、準備します</li><li> ·7H 2 O EBSS、25mMのNaHCO 3を、3 mMの硫酸マグネシウムを含むBSA /アールの平衡塩溶液（EBSS）溶液を調製し、4％BSA、pH7.4中で、先に説明した3。 </li><li> 、150mMのLMEの最終濃度を得MEM培地5mlに純粋LMEを追加することによってLMEを調製する卓上pHメーターを用いてpH 7.4溶液を返し、フィルタ28ミリポリエーテルスルホン（PES）、0.2μmのシリンジを使用して滅菌フィルター。 </li></ol> 3. CGCSを培養<ol><li>以前に3を説明したように4-7日齢のラットの子から小脳を収集します。氷上で5ミリリットル溶液Aを含むペトリ皿に直ちに置きます。 </li><li>シャーレのふたで小脳と場所組織から余分な溶液を捨てます。 </li><li>少なくとも三つの異なる方向によく燃え上がっかみそりの刃で細かく組織をみじん切りに。 </li><li> 5分間37℃の水浴中で溶液（失われた制限された組織を確保するために溶液でペトリ皿をすすぐ）B及び場所にみじん切り組織を加えます。静か分おきに振ります。 </li><li>トリプシン、手ぶれやセントを中和するために、チューブに溶液D（20ミリリットル）を追加5分間、65×gでrifuge。 </li><li>上清を捨てます。 </li><li>直径を小さくする3燃え上がっガラスピペットで十分に溶液C.トリチュレート4mlの（約10回ずつ）で再懸濁し、懸濁液をできるだけ均一になるまで。 </li><li>ゆっくり、ゆっくりとBSA / EBSSの上にこのホモジネートを数滴を追加します。それはBSA / EBSSを通じてシンクを開始した場合は、より多くを粉砕するおよび/またはもう少し溶液Cを追加ホモジネートは、BSA / EBSSの上の層に座ってする必要があります。振らないでください。 5分間100×gでスピン。 </li><li>上清およびMEM培地1mlでペレットを再懸濁し（ソフト）を取り外します。 </li><li> MEM培地500μlの80万細胞/カバーガラスで血球計数器およびプレートを用いて細胞数を計測します。 6％CO 2で37℃のインキュベーターに入れ。 </li><li>培地を翌日（分取した後24〜36時間）を変更します。細胞周期阻害剤のアラビノフラノシルシチジン（のAraC）の10μMを含むMEM培地の溶液を作ります。 <ol><li>それぞれについてカバーガラスにして、再び細胞とトップまでの古い培地を250μlを追加、通常のMEM培地250μlを添加し、細胞を洗浄するために、これを吸引し、残りの部分を吸引、新鮮な24ウェルプレートに古い培地250μlのを保持のAraC含有培地250μlのと。 注意：細胞は、 インビトロ （DIV） で 6日間使用することができます。 </li></ol></li></ol> 4.を選択ミクログリアを枯渇（6 DIVに出演） <ol><li> 150mMのLMEの最終濃度が得られるようにMEM培地の別々のアリコートを5 mlに純粋なLMEを追加します。 </li><li> pHを7.4に解決策を戻し、フィルターは28ミリメートルのポリエーテルスルホン（PES）、0.2μmのシリンジフィルターを用いて滅菌します。 </li><li>コントロール培養のための通常のMEM培地と共に10分間37°Cの水浴中でMEM培地と場所での50mM（2×LME）の濃度に溶液を希釈。 </li><li> CGC培養から半分（250μl）をMEM培地を除去し、37℃で保持します。 </li><li>細胞を処理します、または対照培養のためのLMEのない予め温めたMEM培地（250μl）を、25 mMとウェル濃度を与える1： すなわち 2×LME（250μl）を含むMEM培地で1希釈しました。 </li><li> 1時間、6％CO 2の加湿雰囲気中、37℃で細胞をインキュベートします。 </li><li>洗浄細胞を2回新鮮予め温めたMEM培地中のLME含有培地を除去し、その後、対応するウェルに保持された培地と新鮮予め温めたMEM培地を等量置き換えます。 </li><li> （37℃で6％CO 2の加湿）インキュベーターに文化を戻し、任意のさらなる処理の前に24時間休ませるままにします。 </li></ol>

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The authors declare that they have no competing financial interests.

CGCからミクログリアの成功選択的除去を確実にするために最も重要なステップおよび/または混合培養物は、1）滅菌し、健康CGC文化を維持します。 2）フィルタは、LME含有培地を滅菌し、pHを7.4に溶液を戻します。 3）37に保持CGC媒体とLME含有培地を維持 ヒートショックを回避するため°C; 4）細胞をインキュベーターの外に保持される時間を短縮するために迅速に取り組ん。 <p class=...

人間の脳は、推定850億のニューロンおよびグリア1を含むさらに850億非神経細胞を含みます。過去100年間の大部分のために神経科学者は、ニューロンのための構造的支持を提供した受動支持細胞より少しであることがグリア細胞を信じて、神経細胞集団に主に焦点を当てている - それ故に「グリア」のギリシャ語の語源は、英語に翻訳します「接着剤」。しかし最近では、それは、神経膠細胞の相互作用がはるかに根本的な神経生物学、神経生理学、および多くの神経変性疾患の発生および進行の基本的な側面になる可能性があることがますます明らかになりました。小脳顆粒細胞（CGCS）、ヒトの脳で最も豊富な均質なニューロン集団は、小脳を支配し、その細胞成分の90％以上を占めています。したがって、これらの細胞は、Tのためのモデル系として、in vitroで広範に使用されています彼は、神経発達、機能、および病理2-6の検討します。 しかし、CGC培養はまだ間違いなくかなりの割合でミクログリア及びその他のグリアが含まれています。その結果、推定される種々の細胞治療への直接神経細胞応答を表示するCGCデータは、実際に発生する可能性がある - 一部または全体で - 文化の中で、隣接するグリアの間接的な二次応答から。これを評価するために、我々は、選択的に、L-ロイシンメチルエステル（LME）の助けを借りてCGCニューロン培養物からミクログリアを除去しました。 LMEは、もともと選択マクロファージ7を破壊するために使用lysomotropic剤であり、以降も選択的神経、アストロサイト、および混合膠細胞培養8,9,10からミクログリアを枯渇させるために使用されてきました。 LMEは、リソソームを破砕した後、アポトーシス13,14を引き起こしており、マクロファージおよびミクログリアによって内在化されます。マクロファージおよびミクログリアは、彼らがparticulaであることを引き起こして、リソソームにおける特徴豊富ですLMEの治療に暴露されるとRLY脆弱。このプロトコルは、CGC、その他のグリア/ニューロンの培養系を利用した実験でミクログリアの貢献を把握するための強力な、まだシンプルで簡単な方法を提供します。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Forceps&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4142</td>
			<td>The curved end facilitates removal of the cerebellum&#160;</td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3146</td>
			<td>Straight, sharp point facilitates rodent P4-7 dissection&#160;</td>
		</tr>
		<tr>
			<td>L-leucine methyl ester hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>7517-19-3</td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E7510-500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>27964-99-4</td>
			<td>Coat coverslips 1 day before use&#160;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6414</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-ED1 antibody&#160;</td>
			<td>Abcam</td>
			<td>ab31630</td>
			<td></td>
		</tr>
	</tbody>
</table>

selective depletion of microglia from cerebellar granule cell cultures using l-leucine methyl ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

選択的CGCおよび/または混合培養物からミクログリアを排除するために、この技術の能力を正確に識別し、その周囲の細胞からミクログリアを区別するために、研究者のその後の能力に依存しています。 図1に示されるように、これは、例えば、イソレクチンB4として、マイクログリア特異的細胞マーカーを使用して達成することができる。 図2に示すように、星状細?...

Watch this Scientific Journal Video about Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester at JoVE.com

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

L-ロイシンメチルエステルを使用した小脳顆粒細胞培養物からのミクログリアの選択的枯渇

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

selective-depletion-microglia-from-cerebellar-granule-cell-cultures

Research

JoVE Journal

Neuroscience

8.6K ビュー.  University of Washington. Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature. 

ビデオ: Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

器官小脳文化：アポトーシス課題と検出

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

神経科学

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM染料を用いて培養小脳顆粒神経細胞のシナプス小胞プールの補充の定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脳顆粒ニューロンの遺伝子操作<em&gt;体外</em&gt;と<em&gt;インビボ</em&gt;ニューロンの形態と移行を研究するために、

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

共焦点肉眼検査を使用した生後小脳顆粒細胞の移動のex vivoイメージング

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

最適化されたNを使用して急性脳スライスの準備-メチル-D-glucamine 保護回復法

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

小脳顆粒ニューロン培養におけるFluorescein Diacetate-Propidium Iodide二重染色を用いたニューロン生存率の評価

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

神経細胞死とマウス プライマリ小脳顆粒ニューロンの変性をモデリング

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

免疫組織化学準備 ImageJ を用いた組織の顕微鏡写真からミクログリアの形態の定量化

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

準備や髄脊髄小脳スライス培養の免疫染色

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...