Financial support for the experiments presented in the study was provided by the Marie Heim-V&#246;gtlin program of the Swiss National Science Foundation.
We thank Ms. Johanna Nyffeler for developing a set of modified MATLAB programs for screening for statistically significant differences in real-time cell analyzer&#8217;s data and Dr. Yama Abassi for helpful discussion.

の調製<ol><li> 37℃に設定し、組織培養インキュベーター中、5％CO 2で、リアルタイム細胞分析装置のステーションを配置する。無菌条件下で組織培養フード内のすべての細胞培養処理および薬理学的治療を行う。 </li><li>注：培養のための標準的な条件と比較して異なる温度設定を必要とする神経細胞株は、それに応じて調整されるべきである。弱い接着細胞株について、E-プレート96のウェルの底の金微小電極と細胞の相互作用を容易にするために、コーティング剤を使用する。 </li><li>細胞培養治療のための薬理学的化合物の1,000倍ストック溶液（適切な溶媒中の神経保護剤または第二メッセンジャー経路阻害剤 - ジメチルスルホキシドまたは滅菌水）を準備し、-20℃で保管してください。以下の処理は、車両などの溶媒を使用してください。 </li><li>培養ヒト神経芽細胞腫DMEM / F1におけるSK-N-SH細胞約75％の密度に175cm 2の表面を有する細胞培養フラスコ中、10％ウシ胎児血清（FBS）（増殖培地）を補充した2培地。細胞増殖および細胞毒性に対する応答速度の点で実験との整合性を確認するために、（約10継代の）範囲内の細胞継代数にしてください。低い継代数が好ましい。 </li><li> PBSで付着SK-N-SH細胞層を洗浄し、そして37℃で5分間、0.05％トリプシン-EDTA溶液でトリプシン処理。遠心トリプシンを除去し、5分間170×gで細胞。 10ミリリットルの増殖培地中で細胞を再懸濁。セプター細胞カウンターで細胞を計数し、増殖培地で細胞を希釈して300,000細胞/ mlに細胞数を調整する。 </li></ol> 2めっき及びSK-N-SH細胞の増殖<ol><li> E-プレート96の各ウェルに100μlの増殖培地を追加し、平衡化し、室温で組織培養フード内で30分放置する。 E-PLAを挿入37℃のCO 2インキュベーター内でリアルタイム細胞分析装置局におけるテ96。 </li><li>リアルタイムの細胞分析装置のソフトウェアを起動します。レイアウトページで井戸が実験に含ま選択し、編集ボックスに、細胞型、細胞数、名称および細胞治療のために使用される化学化合物の濃度に関する情報を入力します。 注：このプロトコルの目的のためには、少なくとも4回の反復がそれぞれの治療のために推奨されている。 </li><li>スケジュールソフトウェア]ページでセルインデックスと各ステップのスイープ間の間隔を測定する掃引回数を選択することで、実験に含まれる「手順」を決定する。ステップ1バックグラウンド測定のために予め決められているので、セル·インデックスの96時間、15分ごとに測定し、設定してステップ2」ステップを追加します」を選択します。 注：治療のために、高速な反応速度と効果が期待されている、短い間隔でスイープを設定してください。 </li><li>自動的に所定のステップ1を開始するために[スタート]ボタンをクリックとメディアのバックグラウンドインピーダンスを測定。細胞がウェルに添加されると、この値は自動的に各データポイントでのソフトウェアによって減算される。 </li><li>増殖培地中300,000細胞/ mlの先にステップ1.5で調製した）細胞懸濁液を使用する。細胞毒性/神経保護研究および細胞増殖研究のために15,000細胞/ウェルに良く30,000細胞/。 E-プレートを取り出し、細胞毒性/神経保護研究のためにウェルあたり100μlの細胞懸濁液を追加し、50μlの細胞懸濁液及び細胞増殖研究のために、ウェルあたり50μlの増殖培地を追加します。ウェルあたりの細胞数は経験的に、各細胞株のために最適化される必要がある。 </li><li>静かに、細胞のさえメッキ用のE-プレート96を旋回させる。細胞がウェルの底に均等に落ち着く可能にするために、室温で組織培養フード内で30分間、E-プレート96のままにしておきます。 </li><li>リアルタイムの細胞分析装置ステーションにおいて、E-プレート96を挿入し、[スケジュールp上のステップ2を開始時代。ソフトウェアのセル·インデックス·ページ上でプロットページにあるセルの曲線と生セル·インデックス·データを表示することによって、実験を通してセル·インデックス値を監視します。 </li></ol> 3血清欠乏<ol><li> 24時間細胞をプレーティングした後、実験を一時停止、リアルタイム細胞分析装置ステーションからのE-プレート96を削除します。最終濃度×200に - DMEM / F12無血清培地で1,000倍ストックからセカンドメッセンジャー阻害剤を希釈する。それぞれの経路を阻害するため、細胞毒性を開始する前に、神経毒性/神経保護への関与で30分間、試験される第二メッセンジャー経路1μlの阻害剤で細胞を処理する。ステーションに、E-プレート96を返し、実験細胞指数測定を再開します。 </li><li> 30分後、再び実験を一時停止します。ピペット（またはマルチチャンネルピペット）で、E-プレート96のウェルから完全増殖培地を慎重に除去し、接着細胞層を破壊しないように注意しながらウェルの隅にピペットチップのエッジを導くことができる。 200μl/ウェルの無血清DMEM / F12培地（血清欠乏培地）を追加します。細胞の機械的攪拌を最小限にするために細胞培養培地の迅速な交換を確実。 </li><li>最終濃度×200無血清DMEM / F12培地で1,000倍ストックからそれらの神経保護効果について試験されるべき化合物を希釈する。実験計画に従って各ウェルに彼らの神経保護効果について試験する1μlの化合物および第二メッセンジャー経路1μlの阻害剤を追加します。 </li><li>増殖研究のための細胞では培地を変更しないでください。ウェルあたり1μlの治療および増殖培地で培養を継続し、増殖培地中の最終濃度×200に1,000倍の在庫から、試験する化合物を希釈する。 </li><li>以前に設定した96時間、細胞指数測定と15分ごとに継続して実験を再開します。 </li></ol> 4。データAnalysis <ol><li>神経毒性のために/神経保護データは、「正規化細胞指数」を選択することで、実験間のばらつきを低減し、プロットページの「時間を正規化」するために薬物治療や培地交換前の最後の時点に細胞指数を正常化。 </li><li>興味のある実験条件のためにプロットページで井戸を強調表示し、正規化された細胞指数曲線をプロットする「追加」をクリックする。 、または時間の関数としての正規化されたセル·インデックス·曲線などのセカンドメッセンジャー阻害の神経毒性/神経保護効果の動態を観察します。 </li><li>増殖に対する効果は神経保護/神経毒性作用に関与しているかどうかを評価するために、時間の関数として増殖培地中で試験薬物治療のための細胞指数​​曲線を観察します。 </li><li>結果の統計的評価を開始するために、Excelファイルにセルインデックスソフトウェアのページからすべてのセルインデックス時点の実験情報をエクスポートします<br/&gt;注：統計分析の最初のステップのp値は、ウェルチのt検定を使用して修正されたMATLABプログラムのセットがsemilogarithmicallyプロットした結果、（補助コードファイルとして提供される）時間スケールに対して反転軸を利用している。これらのプログラムは、全体のリアルタイム細胞分析実験の時間経過にわたってp値の検出を可能にするため、より詳細な統計分析のための時間ポイントの選択を支援する。プログラムの詳細については、補足資料を参照してください。 </li><li>特定の時点で異なる処理条件の下で細胞指数値の差の統計学的分析のために、エクスポートされたExcelファイルからの各時点での選択セルのインデックス値。統計ソフトウェアを使用してポストホックテストに続いて、分散の一方向または双方向の分析（ANOVA）を用いて選択された時点におけるセルインデックス値を分析する。 </li></ol>

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現在のビデオ·記事の出版費用は、「ACEA Biosciences社」が主催した。

現在のプロトコルは、連続してラベルを含まない条件下で神経細胞株における化合物の神経保護/神経毒性作用を評価し、効果に関与するセカンドメッセンジャー経路への洞察を得るために、リアルタイム細胞分析装置の有用性を示す。 細胞毒性及び細胞増殖に対する薬物の効果を研究するためのリアルタイム細胞分析の効用が一般的に認識されていても、唯一のいくつ?...

神経細胞死は、多くの脳関連障害1の病態生理学において重要な役割を果たしている。 体外毒性アッセイにおける信頼性と高スループットの利用可能性は、神経毒性のメカニズムにより良い洞察を得るために、薬剤開発の2中の治療候補としての神経保護分子を選択するのに役立つことが重要です。しかし、最も広く速度論的分割を可能としない単一の時点で神経毒性/神経保護を評価するin vitroで神経毒性assays.They で使用するには多くの制限があります。多くの場合、シグナル伝達経路に干渉し、同じ細胞集団においてさらなる研究を制限することができるラベルまたはプローブを使用し、多くの場合、労働集約的であり、多くの場合、機構的洞察を提供しない。本研究では、リアルタイムで及び下神経細胞株における神経毒性および神経保護を決定するために、リアルタイムのインピーダンスベースの細胞分析装置の有用性を実証無標識効果に関与してセカンドメッセンジャー経路の解析を通って下流のメカニズムへの洞察を提供するための条件や。 以前の研究は、細胞毒性、ならびに標準的な技術3,4,5,6と比較して細胞株における細胞増殖への影響を決定するために、リアルタイム細胞分析の妥当性を確認した。例えば、良好な相関が基礎的な増殖条件下で、HeLa細胞における3つの異なる毒性パラダイムの後いくつかの時点で、標準的な細胞生存率WST-1アッセイの読み出し及び細胞指数値の間で観察された。細胞指数の測定によって評価し、標準的に使用されるスルホローダミンB（SRB）アッセイ4時微小管安定化剤パクリタキセル誘発A549およびMDA-MB-231細胞の増殖および細胞毒性に非常に類似した値を示した。不死化海馬ニューロンの神経細胞株では、HT-22細胞指数測定は能力tの検証されたO広く使用3-（4,5 -ジメチルチアゾール2 -イル）2,5 -ジフェニル-ブロミド（MTT）アッセイ5に対する細胞増殖、グルタミン酸細胞毒性および細胞保護作用を検出する。同じ研究において、MTTアッセイの結果とセル·インデックス·測定も汎カスパーゼ阻害剤QVD 5によって成長因子欠乏および細胞毒性の救出後の神経前駆細胞の増殖、細胞毒性を測定する際によく相関していた。バンデタニブ（血管内皮成長因子受容体および上皮成長因子受容体阻害剤）をNIH 3T3細胞に誘導される細胞毒性は、細胞指数値またはニュートラルレッド取り込みアッセイ6で測定し、同様の結果を示した。 私たちは、最近、セロトニン2Aの神経保護効果を評価するために、リアルタイム細胞分析システムを使用した（5-HT 2A）受容体アゴニスト（±）-2,5 -ジメトキシ-4 -ヨードアンフェタミン塩酸塩神経細胞株における（DOI）（ SK-N-SH細胞）およびセコの関与についてスクリーニングNDメッセンジャー経路観察神経保護7上でそれらの化学的阻害の効果をモニタリングを通じて。興味深いことに、5-HT 2A受容体は一般的であり、明確な第二メッセンジャー経路8の両方を活性化することができる（DOIとリスリド、それぞれのような）幻覚やnonhallucinogenic両方アゴニストを持っています。 提示技術の利点は、それが、日の過程で細胞生存に対するリアルタイム情報を収集するために関与するセカンドメッセンジャー経路を描写するために、神経保護の増殖効果の寄与の可能性を評価するために、最適な時間を選択することを可能にすることである同じ細胞集団に対する追加のエンドポイント研究のため。現在のプロトコルでのワークフローの概略図が図1に示されている。

<table border="0" cellpadding="0" cellspacing="0" width="1015">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Name of Material/ Equipment</td>
			<td style="width:161px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:457px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">xCELLigence&#160; RTCA SP system bundle</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 00380601030</td>
			<td>consists of RTCA Analyzer, RTCA SP Station and RTCA Control Unit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">E-plate 96</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 05232368001</td>
			<td>for culturing the cells, inserted in the RTCA SP Station</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">SK-N-SH cells</td>
			<td style="width:161px;">ATCC - (in partnership with LGC Standards)</td>
			<td style="width:119px;">HTB-11</td>
			<td>can be replaced by another adherent neuronal cell line of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMEM/F12&#160;</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8437</td>
			<td>cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">fetal bovine serum</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">16140-063</td>
			<td>supplements proliferation, but not serum deprivation medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">tisue culture flask T175</td>
			<td style="width:161px;">Sarstedt</td>
			<td style="width:119px;">83.1812.302&#160;</td>
			<td>for culturing cells, which will be later plated on the E-Plate 96</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">0.05% Trypsin-EDTA</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">25300-054</td>
			<td>for trypsinization of cells cultured in tissue culture flasks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Scepter 2.0 cell counter</td>
			<td style="width:161px;">Merck Millipore</td>
			<td style="width:119px;">PHCC20060</td>
			<td>automated cell counter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">phosphate-buffered saline</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">10010-015</td>
			<td>for washing the cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">(&#177;)-DOI hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D101</td>
			<td>5-HT2A agonist for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">LY-294,002 hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">L9908</td>
			<td>PI3-K inhibitor for cell culture treatment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">lisuride maleate</td>
			<td style="width:161px;">Tocris Bioscience</td>
			<td align="right" style="width:119px;">4052</td>
			<td>compound with 5-HT2A agonistic activity for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">dimethyl sulfoxide</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4540</td>
			<td>for dissolving LY-294,002 and lisuride maleate</td>
		</tr>
	</tbody>
</table>

real-time impedance-based cell analyzer as a tool to delineate molecular pathways involved in neurotoxicity and neuroprotection in a neuronal cell line

多くの脳関連障害は、それらの病態生理に関与する神経細胞死を持っている。関連する薬剤や下流経路の神経保護または神経毒性作用を研究するためのin vitroモデルの改善は、神経保護/神経毒性の分子機構への洞察を得ることに役立つだろうと、潜在的に医薬品開発を容易にすることができる。しかし、多くの既存のインビトロ毒性アッセイは、主要な制限があります-ほとんどがいない時間経過や効果の動態を観察することができ、単一の時点での神経毒性および神経保護を評価します。また、リアルタイムで神経保護に関与する下流のシグナル伝達経路に関する情報を収集する機会が非常に重要であろう。現在のプロトコルでは、ラベルフリーかつリアルタイムコンディット下の神経細胞株におけるセロトニン2A（5-HT 2Aの）受容体アゴニストの神経保護効果を決定するために、リアルタイムのインピーダンスベースの細胞分析装置の使用を記載インピーダンス測定を用いてイオン。さらに、本発明者らは、第二メッセンジャー経路の阻害剤は、神経保護効果に関与する下流の分子を記述するために使用できることを実証している。また、細胞増殖への影響が観察された神経保護効果に寄与するかどうかを決定するために、この技術の有用性を説明する。システムは、セルのセンサーとして、ウェルの底面上に金微小電極アレイを交互に含んでE-プレートと呼ばれる特殊なマイクロエレクト​​ロニクスプレートを利用する。インピーダンス読み出しは、接着細胞、細胞生存率、形態、および接着の数によって修正される。細胞指数と呼ばれる無次元パラメータは、電気インピーダンス測定値から導出され、セルの状態を表すために使用される。全体的に、リアルタイムのインピーダンスベースの細胞分析装置は、以上に寄与し、リアルタイム、ラベルフリー神経保護及び神経毒性の評価、および第二のメッセンジャー経路の関与の評価を可能にする治療候補を選択するためのin vitroでの潜在的な神経保護化合物の詳細かつ高スループット評価、。

<html> 血清枯渇は、リアルタイム細胞分析装置で連続的に監視することができる細胞指数値を、減少につながる 細胞への神経毒性刺激が提示された技術と、特定の神経毒性刺激と勉強細胞型に依存するのダイナミクスをリアルタイムで監視することができるセルインデックス値の減少につながる。 図2の増加を示しています細胞SK-N-SH細胞を、プラ...

Watch this Scientific Journal Video about Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line at JoV

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

リアルタイム神経細胞株に神経毒性および神経保護に関与する分子経路を描写するツールとしてのインピーダンスベースの細胞分析装置

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

real-time-impedance-based-cell-analyzer-as-tool-to-delineate

Research

JoVE Journal

Neuroscience

11.4K ビュー.  University of Zürich. Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

ビデオ: Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

複数のスケールでデジタル化された神経形態の自動化されたショールの分析

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

神経科学

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト神経前駆細胞と神経細胞の効率的な導出

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

グルタミン酸とGABAの高速マイクロイオン導入：シナプス統合を調査するために便利なツール

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.
Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

神経高密度コア小胞の超解像イメージング

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

神経発火率を操作することによってアストロサイト受容体の可塑性を誘導すること

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

軸索分岐と神経形態形成中に形質膜配信の役割を定義するために組み合わせる方法論を活用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

ナノ粒子神経毒性を決定するために、代理バイオセンサーとしてミクログリア

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

子宮内エレクトロポレーションは、神経部分集団の興奮とシングルセルの接続を研究するためのアプローチ

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

研究保護および腫瘍細胞の侵襲性をモデルとして脊髄海馬スライス培養

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...