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ことです。健康と子犬の生存を保証するには、また氷麻酔時間 (< 4 分) を最小限に抑え、移植手順中に光で加熱した子犬を保つ重要なとしてます。
移植された脳では有意な変動があること、他の脳が何千人も含まれている間いくつかない接木のセルを含めることができます。移植細胞密度を高めるかどちらの可能性が高まるが、接ぎ木の子犬の数を増やすことができます細胞移植、収穫数を増やすことができます解剖のためのいくつかの P0 P2 リットルを組み合わせること成功した移植。適切な脳の領域をターゲットに、子犬の頭が安定し、脳の中にマイクロ ピペットを下げるときに移動しないことが重要です。頭のシフトやマイクロ ピペット シャフトの曲げは、(側脳室に細胞の損失) などの mistargeting や不正確な注射で起因できます。また、(一方的または両側) 子犬あたり複数の注入のサイトを実行すると、1 つの注射部位を mistargeted 場合の成功率が向上します。将来的に子犬を安定化しより正確で一貫性のある注射を提供より効率的な脳定位固定装置戦略を開発するが最適ででしょう。
Th P0 P2 時間枠は、科学的および技術的な理由のために選ばれました。この歳で、ほとんど介在ニューロン前駆体ターミナル サイトに移行しているが、最小限の環境相互作用があります。1 つは、特定の目的のためのこれらの縦長を調整するがありますので、この時間枠は他の種類の神経細胞と脳の領域のことは当てはまらないかもしれない。たとえば、MGE 収穫細胞とこれらの P0 P2 収穫介在ニューロン前駆体の治療の可能性を比較する興味深いだろう: 一つの時代より多くの利点があります。 および/または他のより小さいによって望ましくない副作用。さらに、それは heterochronic 移植 (P0 P2 細胞の収集と P7 10 脳へ、またはその逆を移植など) を実行する面白いかもしれない細胞の運命と成熟に影響可能性があります環境でどのように変化を識別します。P0 2 で注射を実行することの利点は、頭蓋骨が比較的薄く鋭いマイクロ ピペットと容易に侵入することができます。削除または頭蓋骨が薄くなる P5 上任意の注射が必要になります。
このプロトコルで説明する分析脳部位間発現される介在のサブタイプの特定の特性に制限されます。将来的に、1 つは単一のセルの配列の完全移植細胞のトランスクリプトームを特徴づける力を利用することが。この公平な方法は、特定の遺伝子 (または大きいシグナル伝達カスケード) を識別することが、強く濃縮または候補者への洞察力を提供する運命に影響を与える環境の手がかりのコントロールに比べて移植細胞の枯渇決定または成熟します。

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 border="0" cellpadding="0" cellspacing="0" style="width:776px;" width="777">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S7653</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium bicarbonate</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S6297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Potassium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium phosphate monobasic</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S0751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Calcium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">C5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Magnesium chloride</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">M2670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glucose</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">G7528</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sucrose</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">S7903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Brain Matrices</td>
			<td style="width:203px;">Roboz</td>
			<td style="width:136px;">SA-2165</td>
			<td>Only needed if harvesting striatum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fine point Dumont Forceps</td>
			<td style="width:203px;">Roboz</td>
			<td style="width:136px;">RS-4978</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Microdissecting scissors</td>
			<td style="width:203px;">Roboz</td>
			<td style="width:136px;">RS-5940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Razor blades</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12-640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Pasteur pipettes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">1367820C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Nanoject III</td>
			<td style="width:203px;">Drummond</td>
			<td style="width:136px;">3-000-207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Manual Manipulator w/ stand</td>
			<td style="width:203px;">World Precision Instruments&#160;</td>
			<td style="width:136px;">M3301R/M10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">5 ml round bottom plastic tubes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">149591A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">60 mm Petri dishes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12556001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">100 mm Petri dishes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12565100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Pronase</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">10165921001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">16140063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DNase I</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">4716728001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Celltrics 50um filters</td>
			<td style="width:203px;">Sysmex</td>
			<td style="width:136px;">04-0042327</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Trypan blue</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">15-250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Hemocytometer</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">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 間介在ニューロン前駆体移植を受けた脳に収穫され。これらのタイプの試金は通常マウスで、P21 P30 の間しばしば行わが、移植細胞の成熟は郭清/分離手続により若干遅れる可能性があります、追加の 5-10 日を待ってお勧めこれを補うために遅延の成熟。実行する解析のタイプは、脳を収穫する適切な戦略を左右します。特に、我々 は、または特定のサブタイプ20に対してバイアスが特定の介在のサブグループの優遇細胞死を観察しなかった。
免疫組織化学的解析, 4% パラホルムアルデヒドで灌流固定マウスと脳が削除されました。50 μ m vibratome スライスは、不凍液に格納されていることや前述20として是非、処理対象脳領域を通じて準備されたいた。いくつかの脳には、不適切な対象化 (例えば、心室に余りに深く注入)、失われた細胞またはホストによって移植された細胞の拒絶や移植術の中にアポトーシスを受ける可能性があります任意のトマト + 細胞が含まれていませんでした。推定されている最後のセルのカウントに基づいて、20、その他移植手順22,23沿ったものですが、移植細胞の唯一の 2-5% に生き残る。
当然のことながら、トマト + 成功移植、数十から数千のトマト + 細胞 (図 4 a) に至るまでのセルの合計数に大きな変動があった。移植細胞にローカライズされた正しい地域、多くの介在ニューロンの形態と特徴介在神経化学的マーカー (図 4 b) を表示します。似たような細胞生存率の数字と成熟のプロファイルは、細胞が異所性移植 (図 4) で新しい環境に接木されたときも観察されました。
免疫組織化学的解析に加えて彼ら脳回路に統合されているし、期待される本質的な発火特性を表示ことを確認移植細胞の電気生理学的解析を行った。P30 35 マウスから収穫された脳、スライス以前として生理学的録音準備説明20。接木の介在ニューロンが大人のような生理学的性質を提示し、パターン可能性があります異なる焼成の特徴をよく特徴付けられる介在ニューロンの代表サブタイプ (図 5 a)、ことを示唆する接ぎ木介在ニューロン正しくホスト環境で成熟することができた。移植細胞が神経ネットワークに統合されたことを確認するため sEPSCs がまた記録された (図 5 b)。また、錐体細胞から記録する近くローカライズされた移植介在ニューロンによって続いて ChR2 を表現する介在ニューロンの移植のサブセットを行った。これらのデータは、gaba 作動性シナプス電流は青い光 (図 5-D) によって誘発されことを示した。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57723/57723fig4.jpg"> 図 4: 接木介在ニューロン前駆体トマト + P1 で前駆体介在ニューロンをホスト脳移植された P30 WT マウスから代表的なセクションに設定します。(A)つながれた皮質-皮質の移植、移植細胞皮質のすべてのレイヤーを読み込む、内因性介在ニューロンを模した形態を表示します。選択した画像は、セクションでは右側に移植と比較してはるかに大きいトマト + 細胞数を持つ左の画像と別の移植から細胞数の変動を強調表示します。(B)低倍率 (左) とつながれた海馬・海馬移植から高倍率 (右) 代表的なセクション。地層属三角骨 (SP) トマト + セル表現 PV (可能性が高いバスケット細胞)、内因性海馬介在ニューロンと同様に対しトマト + 地層オリエンスの細胞の大半が (そう) SST (可能性が高い O LM 細胞) を表現に注意してください。トマト + 線条体に存在に異所性移植 (皮質・線条体) の(C)の例です。スケール バー = B、C で 50 μ m と B のハイパワー等の低倍率パネル A の 200 μ m<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57723/57723fig4large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57723/57723fig5.jpg"> 図 5: 接木介在ニューロン生理成熟し、ニューロン ネットワークのホストの統合 (A)最高の発火周波数の代表例は、接木介在ニューロンから記録されます。左股関節-Ctx 移植から高速スパイキング介在注入現在手順:-100 pA と 520 pA;右Ctx-Ctx 移植から非高速スパイキング介在ニューロン注入現在手順:-100 pA と 360 ペンシルバニア州(B) sEPSCs の例は、ヒップに腰を移植から遅くスパイキング介在ニューロンに記録されます。(C)代表的な画像表示Nkx2.1-Cre;Ai32 biocytin いっぱい (赤) 錐体細胞と移植細胞 (YFP) Ctx に Ctx から。スケールバー = 50 μ m。(D)青色光パルスによって誘発される gaba 作動性シナプス電流の例は記録 [145 mM] Cl - 錐体細胞に記録。黒で平均トレース;赤で Gabazine の存在下で平均応答が記録されます。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57723/57723fig5large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

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

Nanyang Technological University

Research

JoVE Journal

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

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

ウイルスベースのCre組換えによる出生後のマウスの脳の発達における遺伝子機能のモザイク解析

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

神経科学

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

神経向性マウス肝炎ウイルスに感染したマウスの脊髄にマウス神経幹細胞の外科的移植

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

共焦点光シート顕微鏡全体マウスの脳のミクロンスケールの分解能光トモ

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

生体産後エレクトロポレーションおよびマウスの急性脳スライスにおける神経芽細胞移動のタイムラプスイメージング中

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

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 ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

血液-脳関門の高分解能共焦点イメージング: イメージング、三次元、トランスサイトーシスの定量化

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生児マウス脳の線条体細胞の遺伝的操作の定位手術

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

Γ-アミノ酪酸受容体をモジュレート新規化合物の探索の方法を入力、神経伝達

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

複数の齧歯動物の頭脳の同時セクショニング

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

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 ...