この研究は、健康の国民の協会 (K99MH104595) および T.J.P. に発育研究所所内研究プログラムに支えられました。 そのラボでこのアプローチは創業 Gord Fishell に感謝いたします。

<ol>
	<li>Bozzi, Y., Casarosa, S., Caleo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epilepsy+as+a+neurodevelopmental+disorder.">Epilepsy as a neurodevelopmental disorder.</a> Front Psychiatry. 3 (19), (2012).</li><li>Takano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneuron+Dysfunction+in+Syndromic+Autism:+Recent+Advances.">Interneuron Dysfunction in Syndromic Autism: Recent Advances.</a> Dev Neurosci. , (2015).</li><li>Inan, M., Petros, T. J., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Losing+your+inhibition:+linking+cortical+GABAergic+interneurons+to+schizophrenia.">Losing your inhibition: linking cortical GABAergic interneurons to schizophrenia.</a> Neurobiol Dis. 53, 36-48 (2013).</li><li>Batista-Brito, R., Machold, R., Klein, C., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+in+cortical+interneuron+precursors+is+prescient+of+their+mature+function.">Gene expression in cortical interneuron precursors is prescient of their mature function.</a> Cereb Cortex. 18 (10), 2306-2317 (2008).</li><li>Flames, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delineation+of+multiple+subpallial+progenitor+domains+by+the+combinatorial+expression+of+transcriptional+codes.">Delineation of multiple subpallial progenitor domains by the combinatorial expression of transcriptional codes.</a> J Neurosci. 27 (36), 9682-9695 (2007).</li><li>Wonders, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spatial+bias+for+the+origins+of+interneuron+subgroups+within+the+medial+ganglionic+eminence.">A spatial bias for the origins of interneuron subgroups within the medial ganglionic eminence.</a> Dev Biol. 314 (1), 127-136 (2008).</li><li>Inan, M., Welagen, J., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+bias+in+the+mitotic+origins+of+somatostatin-+and+parvalbumin-expressing+interneuron+subgroups+and+the+chandelier+subtype+in+the+medial+ganglionic+eminence.">Spatial and temporal bias in the mitotic origins of somatostatin- and parvalbumin-expressing interneuron subgroups and the chandelier subtype in the medial ganglionic eminence.</a> Cereb Cortex. 22 (4), 820-827 (2012).</li><li>Petros, T. J., Bultje, R. S., Ross, M. E., Fishell, G., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical+versus+Basal+Neurogenesis+Directs+Cortical+Interneuron+Subclass+Fate.">Apical versus Basal Neurogenesis Directs Cortical Interneuron Subclass Fate.</a> Cell Rep. 13 (6), 1090-1095 (2015).</li><li>Taniguchi, H., Lu, J., Huang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spatial+and+temporal+origin+of+chandelier+cells+in+mouse+neocortex.">The spatial and temporal origin of chandelier cells in mouse neocortex.</a> Science. 339 (6115), 70-74 (2013).</li><li>Bandler, R. C., Mayer, C., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+interneuron+specification:+the+juncture+of+genes,+time+and+geometry.">Cortical interneuron specification: the juncture of genes, time and geometry.</a> Curr Opin Neurobiol. 42, 17-24 (2017).</li><li>Kepecs, A., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneuron+cell+types+are+fit+to+function.">Interneuron cell types are fit to function.</a> Nature. 505 (7483), 318-326 (2014).</li><li>Mayer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+diversification+of+cortical+inhibitory+interneurons.">Developmental diversification of cortical inhibitory interneurons.</a> Nature. 555 (7697), 457-462 (2018).</li><li>Mi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+emergence+of+cortical+interneuron+diversity+in+the+mouse+embryo.">Early emergence of cortical interneuron diversity in the mouse embryo.</a> Science. 360 (6384), 81-85 (2018).</li><li>Alvarez-Dolado, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+inhibition+modified+by+embryonic+neural+precursors+grafted+into+the+postnatal+brain.">Cortical inhibition modified by embryonic neural precursors grafted into the postnatal brain.</a> J Neurosci. 26 (28), 7380-7389 (2006).</li><li>Baraban, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+seizures+by+transplantation+of+cortical+GABAergic+interneuron+precursors+into+Kv1.1+mutant+mice.">Reduction of seizures by transplantation of cortical GABAergic interneuron precursors into Kv1.1 mutant mice.</a> Proc Natl Acad Sci U S A. 106 (36), 15472-15477 (2009).</li><li>De la Cruz, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneuron+progenitors+attenuate+the+power+of+acute+focal+ictal+discharges.">Interneuron progenitors attenuate the power of acute focal ictal discharges.</a> Neurotherapeutics. 8 (4), 763-773 (2011).</li><li>Gilani, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneuron+precursor+transplants+in+adult+hippocampus+reverse+psychosis-relevant+features+in+a+mouse+model+of+hippocampal+disinhibition.">Interneuron precursor transplants in adult hippocampus reverse psychosis-relevant features in a mouse model of hippocampal disinhibition.</a> Proc Natl Acad Sci U S A. 111 (20), 7450-7455 (2014).</li><li>Larimer, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caudal+Ganglionic+Eminence+Precursor+Transplants+Disperse+and+Integrate+as+Lineage-Specific+Interneurons+but+Do+Not+Induce+Cortical+Plasticity.">Caudal Ganglionic Eminence Precursor Transplants Disperse and Integrate as Lineage-Specific Interneurons but Do Not Induce Cortical Plasticity.</a> Cell Rep. 16 (5), 1391-1404 (2016).</li><li>Martinez-Cerdeno, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+MGE+precursor+cells+grafted+into+adult+rat+striatum+integrate+and+ameliorate+motor+symptoms+in+6-OHDA-lesioned+rats.">Embryonic MGE precursor cells grafted into adult rat striatum integrate and ameliorate motor symptoms in 6-OHDA-lesioned rats.</a> Cell Stem Cell. 6 (3), 238-250 (2010).</li><li>Quattrocolo, G., Fishell, G., Petros, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+Transplantations+Reveal+Environmental+Influences+on+Interneuron+Diversity+and+Maturation.">Heterotopic Transplantations Reveal Environmental Influences on Interneuron Diversity and Maturation.</a> Cell Rep. 21 (3), 721-731 (2017).</li><li>Xu, Q., Tam, M., Anderson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+Nkx2.1-lineage+cells+in+the+mouse+telencephalon.">Fate mapping Nkx2.1-lineage cells in the mouse telencephalon.</a> J Comp Neurol. 506 (1), 16-29 (2008).</li><li>Thompson, L., Bjorklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival,+differentiation,+and+connectivity+of+ventral+mesencephalic+dopamine+neurons+following+transplantation.">Survival, differentiation, and connectivity of ventral mesencephalic dopamine neurons following transplantation.</a> Prog Brain Res. 200, 61-95 (2012).</li><li>Liang, Y., Agren, L., Lyczek, A., Walczak, P., Bulte, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+progenitor+cell+survival+in+mouse+brain+can+be+improved+by+co-transplantation+of+helper+cells+expressing+bFGF+under+doxycycline+control.">Neural progenitor cell survival in mouse brain can be improved by co-transplantation of helper cells expressing bFGF under doxycycline control.</a> Exp Neurol. 247, 73-79 (2013).</li></ol>

著者が明らかに何もありません。

このプロトコルの 1 つの重要な側面は、細胞の生存を最大にします。組織や細胞が確実常に氷冷 carboxygenated sACSF では、細胞の生存を促進するために必要です。効率的な解離と解離細胞脳環境の内外で様々 なソリューションを過ごす時間の長さを最小限に抑える戦略が必要です。脳解剖され、移植の数、に応じて実験の長さを減少する郭清および/または移植の手順で支援パートナーに有益な?...

適切な皮質機能は、興奮性の投射ニューロンと介在ニューロン gaba 作動性抑制性、明瞭な形態、電気生理学的特性、接続性と非常に異質人口のバランスを必要とする神経マーカー。異常な開発および介在ニューロン (および特定の介在サブグループ) の機能は、統合失調症、自閉症、てんかん1,2,3などの精神疾患の病態にリンクされています。さらに、これらの脳障害に関与する多くの遺伝子を若い介在4で濃縮され強く。したがって、通常の開発と多くの脳疾患の潜在的な病因を理解する介在ニューロンの運命決定と成熟を調節するメカニズムの理解が必要です。
2 一時的な萌芽期の構造、内側と仙骨神経節隆起から主に前脳のニューロンが生まれて (MGE と CGE、それぞれ)。これらの後の細胞 (介在ニューロン前駆体) さまざまな回路に組み込まれ、終脳全体で分散する長引く接線方向の移動相を受けます。MGE 派生介在ニューロンから成る 3 主重複、神経に定義されたサブグループ: 高速パルブアルブミン (PV+) 介在ニューロン、高速ソマトスタチン (SST+) 介在ニューロンのスパイクをスパイクと後期神経スパイク硝酸neurogliaform とアイビーの海馬の細胞を構成する酸化物合成酵素 (nNOS+) の介在。多数の研究所が太陽光発電+または SST + 介在ニューロン、モルフォゲン、介在ニューロン前駆体の生年月日および神経因性部モードの空間勾配を含む初期の運命の決定を調整する、MGE 内のいくつかのメカニズムを識別しました。5,6,7,8,9,10. 介在ニューロンが当初 '基本クラス' に区別して徐々 に成熟して '決定的なクラス'11彼らの環境と対話することが提案されています。最近の証拠いくつかの成熟した介在のサブタイプことを示して遺伝子ハードワイ ヤードこれらの細胞は、神経節隆起の後になると初期定義された固有の遺伝的プログラムが以前よりも大きな役割を果たすことを示す感謝12,13。ただし、固有の遺伝的プログラムは、異なる介在サブタイプにドライブ分化する環境手がかりと対話する方法の重要な問題は大部分は未踏。
数多くの研究が様々 な成熟した細胞を移植し、一般的にローカルの内因性回路14,15を抑制する GABA をリリース コンセンサス結果による脳領域に直接 MGE の萌芽期細胞を移植します。 16,17,18,19。ひと誘導多能性幹細胞 (こと) を使用して大きな関心を生成している観測を約束これらの様々 な脳の病気を治療するために介在を派生します。ただし、これらの研究のいくつかの非常には、これらの移植細胞成熟成熟したニューロンの型にかを評価するときに、1 つの重要なコンポーネントは、トランスレーショナル アプローチについて考えています。
環境がどのように介在ニューロン分化と成熟に影響を与えるに対処するため接木介在ニューロンは、ホストの機能を採用するかどうかを調べるために新しい脳環境に未熟な介在ニューロン前駆体を移植して戦略を考案環境またはドナー環境20から機能を保持します。MGE の移植は、MGE は介在および多数の脳領域21全体分散 gaba 作動性投射細胞の混合された人口を含んでいるのでこの質問に対処するため適していません。これらの MGE の細胞は移行が知らなくても、1 つない完全に評価できますこれらの移植、脳環境によってどのように影響を受けるのか。移行を完了し、自分の目標に達している未熟な細胞を取得して初期産後縦長で介在ニューロン前駆体を収穫してこの問題を回避の頭脳領域しますが、最小限の操作環境で。大脳皮質の異なる領域間発現ニューロンの特定の機能に焦点を当て、その後、ホスト環境が介在プロパティを変更する方法を決定できます。このプロトコルで説明する一般的なアプローチは、任意の捜査に適用されるべき新しい環境で挑戦したときを動作することをどのように若いニューロンを検討したいです。

<table><tbody><tr><td>Sodium chloride</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S6297</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>S0751</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C5080</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma</td><td>M2670</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G7528</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S7903</td><td></td></tr><tr><td>Brain Matrices</td><td>Roboz</td><td>SA-2165</td><td>Only needed if harvesting striatum</td></tr><tr><td>Fine point Dumont Forceps</td><td>Roboz</td><td>RS-4978</td><td></td></tr><tr><td>Microdissecting scissors</td><td>Roboz</td><td>RS-5940</td><td></td></tr><tr><td>Razor blades</td><td>ThermoFisher</td><td>12-640</td><td></td></tr><tr><td>Pasteur pipettes</td><td>ThermoFisher</td><td>1367820C</td><td></td></tr><tr><td>Nanoject III</td><td>Drummond</td><td>3-000-207</td><td></td></tr><tr><td>Manual Manipulator w/ stand</td><td>World Precision Instruments&amp;nbsp;</td><td>M3301R/M10</td><td></td></tr><tr><td>5 ml round bottom plastic tubes</td><td>ThermoFisher</td><td>149591A</td><td></td></tr><tr><td>60 mm Petri dishes</td><td>ThermoFisher</td><td>12556001</td><td></td></tr><tr><td>100 mm Petri dishes</td><td>ThermoFisher</td><td>12565100</td><td></td></tr><tr><td>Pronase</td><td>Sigma</td><td>10165921001</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher</td><td>16140063</td><td></td></tr><tr><td>DNase I</td><td>Sigma</td><td>4716728001</td><td></td></tr><tr><td>Celltrics 50um filters</td><td>Sysmex</td><td>04-0042327</td><td></td></tr><tr><td>Trypan blue</td><td>ThermoFisher</td><td>15-250-061</td><td></td></tr><tr><td>Hemocytometer</td><td>ThermoFisher</td><td>02-671-6</td><td></td></tr></tbody></table>

homochronic transplantation of interneuron precursors into early postnatal mouse brains

神経細胞の運命決定と成熟には、遺伝的プログラムや環境の信号間の複雑な相互作用が必要です。しかし、この分化過程を規制する外因性のメカニズムと組み込みのロールを disentangling はすべて発達にかの難問です。この問題は、gaba 作動性介在ニューロン、一時的な萌芽期の構造から生まれた、終脳全体を分散させるため長引く渡り鳥先行き非常に異種細胞集団の拡大です。どのように別の脳環境影響介在ニューロン運命と成熟を探索するには、新生児マウス (P0 P2) で特定の脳領域からの蛍光に分類された未熟な介在ニューロン前駆体を収穫するためのプロトコルを開発しました。この歳で、介在ニューロンの移行がほぼ完了してこれらの細胞は比較的小さなシナプス統合の最終的な休息環境に居住しています。フローサイトメトリーによって単一のセル、ソリューションのコレクション、次のこれらの介在ニューロン前駆体は P0 P2 野生型産後子犬に移植しました。両方つながれたを実行することによって (例えば、皮質-皮質) または異所性 (例えば、野-海馬) 移植、1 つの缶が彼らの運命, 成熟, および回路の集積の影響新しい脳環境で未熟な介在に挑戦を評価。脳が成体マウスの収穫できアッセイはさまざまな移植細胞の免疫組織化学的, を含む posthoc 分析、電気生理学的および転写プロファイリングします。この一般的なアプローチは、どのように異なる脳環境を分析するための戦略を持つ研究者がニューロンの開発の多くの側面に影響を与えるし、特定の神経細胞の特性、主にハードワイ ヤード遺伝プログラムによって駆動されている場合を識別または環境の手がかり。

このプロトコルが初期生後脳 (図 1-2) から特定の脳領域を収穫、介在ニューロン前駆細胞の単一細胞状態を収集し、素朴な WT の様々 な脳領域にこれらの細胞を移植する方法を示します出生後の子犬 (図 3)。Posthoc 解析、細胞の形態、神経化学的マーカーおよび電気生理学的特性を特徴付ける P30 35 間介在ニュー?...

Watch this Scientific Journal Video about Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains at JoVE.com

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

新しい脳領域の若いニューロンに挑戦と、神経細胞の運命と成熟の環境の sculpts に重要な洞察を明らかにできます。このプロトコルを記述する特定の脳領域からの介在ニューロン前駆体を収穫し、それらのいずれかの homotopically を移植する手順や出生後の子犬の脳にあり。

早期の生後早期のマウス脳に介在ニューロン前駆体の Homochronic 移植

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

homochronic-transplantation-interneuron-precursors-into-early

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Neuroscience

7.8K ビュー.  Norwegian University of Science and Technology. 新しい脳領域の若いニューロンに挑戦と、神経細胞の運命と成熟の環境の sculpts に重要な洞察を明らかにできます。このプロトコルを記述する特定の脳領域からの介在ニューロン前駆体を収穫し、それらのいずれかの homotopically を移植する手順や出生後の子犬の脳にあり。

ビデオ: Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

生化神経建築研究における工学的神経前駆体の脳内移植

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

遺伝学

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

新生児の脳室下帯のエレクトロ

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

神経科学

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

発生中のマウス胚性皮質における有糸分裂のライブイメージング

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

マウス胚性幹細胞の皮質介在ニューロン前駆細胞への分化

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

萌芽期の神経を研究するためのモデルとしてのマウス脳

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

化学遺伝子解析で作られた皮質間ニューロン前駆体を早期産後マウス脳に移植

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

マウス脳におけるヒト神経前駆細胞の脳内移植と 生体 内生物発光追跡

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using interneurons to replace or mitigate the effects of altered neuronal circuits is an attractive therapeutic avenue. To this end, more knowledge is needed about how human stem cell-derived GABAergic interneuron-like cells (hdINs) mature, integrate, and function over time in the host circuitry. Of particular importance in neurodevelopmental disorders is a better understanding of whether these processes in transplanted cells are affected by an evolving and maturing host brain. The present protocol describes a fast and highly efficient generation of hdINs from human embryonic stem cells based on the transgenic expression of the transcription factors Ascl1 and Dlx2. These neuronal precursors are transplanted unilaterally, after 7 days in vitro, to the hippocampus of neonatal 2-day-old mice. The transplanted neurons disperse in the ipsi- and contralateral hippocampus of a mouse model of cortical dysplasia-focal epilepsy syndrome and survive for up to 9 months after transplantation. This approach allows for investigating the cellular identity, integration, functionality, and therapeutic potential of transplanted interneurons over an extended time in developing healthy and diseased brains.

Transplantation of human pluripotent stem cell-derived GABAergic neurons generated by neuronal programming could be a potential treatment approach for neurodevelopmental disorders. This protocol describes the generation and transplantation of human stem cell-derived GABAergic neuronal precursors into the brains of neonatal mice, allowing long-term investigation of grafted neurons and evaluation of their therapeutic potential.

ヒト幹細胞由来GABA作動性ニューロンの生後早期マウス海馬への移植による神経発達障害緩和

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using ...

発生中のマウス脳における神経細胞およびグリア細胞の動態の可視化

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

著者スポットライト:発達中の脳における細胞移動と遺伝子の役割の探索

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

マウス胚脳切片における移動するニューロンとグリア前駆細胞のタイムラプスイメージング

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

早期の生後早期のマウス脳に介在ニューロン前駆体の Homochronic 移植

要約

さらに動画を探す

この動画の章

生化神経建築研究における工学的神経前駆体の脳内移植

新生児の脳室下帯のエレクトロ

発生中のマウス胚性皮質における有糸分裂のライブイメージング

マウス胚性幹細胞の皮質介在ニューロン前駆細胞への分化

萌芽期の神経を研究するためのモデルとしてのマウス脳

化学遺伝子解析で作られた皮質間ニューロン前駆体を早期産後マウス脳に移植

マウス脳におけるヒト神経前駆細胞の脳内移植と生体内生物発光追跡

ヒト幹細胞由来GABA作動性ニューロンの生後早期マウス海馬への移植による神経発達障害緩和

マウス胚脳切片における移動するニューロンとグリア前駆細胞のタイムラプスイメージング

マウス胚脳切片における移動するニューロンとグリア前駆細胞のタイムラプスイメージング

早期の生後早期のマウス脳に介在ニューロン前駆体の Homochronic 移植

要約

さらに動画を探す

この動画の章

生化神経建築研究における工学的神経前駆体の脳内移植

新生児の脳室下帯のエレクトロ

発生中のマウス胚性皮質における有糸分裂のライブイメージング

マウス胚性幹細胞の皮質介在ニューロン前駆細胞への分化

萌芽期の神経を研究するためのモデルとしてのマウス脳

化学遺伝子解析で作られた皮質間ニューロン前駆体を早期産後マウス脳に移植

マウス脳におけるヒト神経前駆細胞の脳内移植と 生体 内生物発光追跡

ヒト幹細胞由来GABA作動性ニューロンの生後早期マウス海馬への移植による神経発達障害緩和

マウス胚脳切片における移動するニューロンとグリア前駆細胞のタイムラプスイメージング

マウス胚脳切片における移動するニューロンとグリア前駆細胞のタイムラプスイメージング

マウス脳におけるヒト神経前駆細胞の脳内移植と生体内生物発光追跡