この研究プロジェクトは、ARRAサプリメント、5 R01 NS056380によってサポートされていました。追加のサポートは、ウイルスベクターコアとエモリー神経NINDSコア設備助成、P30NS055077の顕微鏡コアを介して提供されました。 SSTR3-GFPレンチウイルスコンストラクトのために、ブラッドリーYoder発信;安定SSTR3-GFP IMCD3細胞ラインのグレッグPazour、我々はGENSATからマウスラインを導出するためのコアをターゲットエモリートランスジェニックマウスと遺伝子に感謝します。モノクローナル抗体は、NICHDの後援の下で開発された発達研究ハイブリドーマ銀行、、から入手し、アイオワ大学、生物科学科、アイオワシティ、アイオワ52242によって維持された。すべての動物の手順はIACUCとエモリー大学のバイオセーフティ委員会によって承認された。

成体マウスは、機械頸椎脱臼により安楽死させる。すべての動物の手順はIACUCとエモリー大学のバイオセーフティ委員会によって承認された。 1。胚の生成<ol><li>クロスタモキシフェン誘導性Creをライン、CAGGCreER、およびdsRedCreレポーターライン点（Tg（CAG-Bgeo、-DsRedを* MST）1Nagy）（ 図1）5,6。娘細胞、クロスCAGGCreERとOlig2-EGFP BACトランスジェニックマウス（Tgは（Olig2-EGFP）EK23Gsat）（ 図1）7,8とDsRedのラインにおけるShhの応答の相対的なタイミングを監視する。 </li><li> 100％エタノールでタモキシフェンを溶解し、Creの発現と細胞の小さなサブセットでDsRedのラベルを誘導するために胚6.5（E6.5）で妊娠した女性に体重40グラムあたり2.5 mgのタモキシフェンの腹腔内注射を行う。 </li><li> 48時間、後、胚を解剖蛍光顕微鏡を用いたDsRed肯定胚を特定し、培養皿にそれらを転送しobservex vivoでの撮影時の電子の単一細胞分裂（下記参照）。 </li></ol> 2。全体マウス胚培養<ol><li> 10％新生仔ウシ血清及び1％ペニシリン/ストレプトマイシン（P / S）9を補充したDMEM/F12（1:1）を含む予め温めた洗浄媒体でE8.5胚を解剖する。 </li><li>直接蛍光顕微鏡下で37℃の加熱ステージ上で解剖、場所後の胚及びGFPおよび/またはDsRedをプラス（下記参照）として識別します。 </li><li> L-グルタミン、フェノールレッドを含まない50％のSprague-Dawley系雄性ラットの血清および50％DMEM/F12（1:1）を含む予め平衡培地500μlの降下との1％に2胚まで転送0.85％NaCl及びP / S 9の1M HEPES。 </li><li>蒸発を防ぎ、培養皿を37℃、5％CO 2インキュベーターに胚を含む転送する媒体を介して平衡化した軽油の薄い層（0.1センチ）を適用します。 </li></ol> 3。ウイルス感染<ol><li>神経上皮におけるラベル繊毛するためには、E8.5胚を含む培養培地500μlの平衡化の一滴までSSTR3-GFPレンチウイルスの5-10μlを、約200万ビリオンを追加します。 </li><li>文化の18時間後、ウイル​​スを洗い流すと、神経管のスライスを準備するために洗浄媒体の数滴に感染した胚を移す。 </li></ol> 4。ライブイメージングのための神経管のスライス標本<ol><li> 1％寒天コーティングされた皿に、マイクロナイフの大きさ0.025ミリメートルを使用して予め温めておいた洗浄媒体におけるE8.5胚の神経管を解剖。 </li><li> 35ミリメートルポリ-L-リジンコートガラスボトムディッシュ上のフェノールレッドなしの平衡化培地150μlのドロップで孤立神経管腹側を下に置きます。 </li><li>するために、ガラスカバースリップの狭い部分で100％純粋なワセリンとマウント神経管の周りに溶けたろうそくのろう（IKEAからろうそく）、ゆっくりと押しから作られた1:1の混合物の少量を入れてサンプルを固定。 </li><li>平衡化した軽油の薄い層（〜0.1センチメートル）（ 図2）皿をカバーしています。 </li></ol> 5。ライブイメージングとタイムラプス共焦点顕微鏡<ol><li>温度を調節する環境室を装備ニコンA1Rレーザ走査共焦点倒立顕微鏡下に置く皿は、37℃、5％CO 2を設定してください。 </li><li> 40倍の油浸対物レンズの使用がOlig2-GFPとDsRedの陽性細胞を監視する一方、GFPラベル繊毛とDsRedの陽性細胞を記録するために60倍の油浸対物レンズを使用してください。 </li><li>タイムラプス撮影条件を設定するためにNISの要素ソフトウェアを開きます。 10分ごとに1.5ミクロン（40倍目標）と0.4μmの（60倍目標）の間隔で最大8ミクロンの間隔で25ミクロンまでのzスタックを取得する。必要に応じて488と561 nmの励起波長と送信チャネルを使用してください。 512×512のサイズで画像を取得する。 int型の複数のユーザ定義領域を設定する同時記録​​を行うことerest。 、スキャン速度1、線平均2とピンホールの大きさ（61、画像のレーザパワーと明るさに最適な露出、使用低いレーザー強度（4.5％までEGFP 488分の405 mCherry 561は最大11％）で調整したDsRedのためのミクロン; Olig2ための28.1ミクロン; SSTR3GFPための72ミクロン、DsRedのとSSTR3GFPための61.3ミクロン、DsRedのとOlig2用58ミクロン）。遺伝的に符号化された蛍光レポーターの使用は、低レーザパワーと明るさを検出することができました。 </li><li> Imaris 3D再構築ソフトウェアを使用して記録されたデータを分析します。 </li></ol> 6。免疫蛍光<ol><li>ガラス皿で1時間氷上で4％パラホルムアルデヒド/ 0.1 Mリン酸緩衝液（4℃）で胚あるいは孤立神経チューブを固定します。 </li><li>氷（4℃）でPBSのサンプル2時間（変化PBS数回）洗浄し、一晩または胚シンクまで、30％スクロース/ 0.1Mリン酸緩衝液に入れ。 </li><li> -80でドライアイスや店舗℃の上、10月に凍結を埋め込むPクライオスタット（10ミリメートル）で切片erform。 </li><li> 1％の熱不活化羊血清および0.1％トリトンX-100 PBS中を含む洗浄溶液中で10分間スライドを洗浄します。 </li><li>ウサギ抗血清Arl13b 1:1500及びマウスモノクローナル抗Arl13b 1時05分（295B/54）;、ウサギ抗Olig2ラットモノクローナル抗RFP（5F8）、1:200以下の濃度で洗浄溶液中の一次抗体を希釈1:300、マウスモノクローナルPax6の、シーッとNkx2.2-すべて1時10分、およびKi67の1:500ポリクローナルウサギ。乾燥避け、4℃で一晩のままにフラット加湿チャンバー内のスライドあたり150μlの、パラフィルムでカバー周り追加℃に</li><li>三回、20分毎に時間室温で洗浄溶液中でスライドを洗浄します。 </li><li> 1:200濃度の洗浄溶液で二次抗体アレクサフルーア488、568、350を希釈。核を染色するためにヘキスト1:3,000またはTO-PRO-3 1:500を使用します。スライドあたり150μlを添加し、光から保護加湿チャンバー内で室温で1時間のままにしておきます。 </li><li>洗浄ソリューションTWのスライドを洗うoが時間、室温で30分間、毎回。 </li><li> 80％でマウントスライドは24時間以内にゴールドアンチフェード試薬およびビューを延ばす。 </li></ol>

<ol>
	<li>Yamada, T., Pfaff, S. L., Edlund, T., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+pattern+in+the+neural+tube:+motor+neuron+induction+by+diffusible+factors+from+notochord+and+floor+plate.">Control of cell pattern in the neural tube: motor neuron induction by diffusible factors from notochord and floor plate.</a> Cell. 73, 673-686 (1993).</li><li>Ericson, J., Muhr, J., Placzek, M., Lints, T., Jessell, T. M., Edlund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+induces+the+differentiation+of+ventral+forebrain+neurons:+A+common+signal+for+ventral+patterning+within+the+neural+tube.">Sonic hedgehog induces the differentiation of ventral forebrain neurons: A common signal for ventral patterning within the neural tube.</a> Cell. 81, 747-756 (1995).</li><li>Briscoe, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeodomain+Protein+Code+Specifies+Progenitor+Cell+Identity+and+Neuronal+Fate+in+the+Ventral+Neural+Tube.">Homeodomain Protein Code Specifies Progenitor Cell Identity and Neuronal Fate in the Ventral Neural Tube.</a> Cell. 101, 435-445 (2000).</li><li>Berbari, N. F., Johnson, A. D., Lewis, J. S., Askwith, C. C., Mykytyn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+ciliary+localization+sequences+within+the+third+intracellular+loop+of+G+protein-coupled+receptors.">Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors.</a> Mol. Biol. Cell. 19 (4), 1540-1547 (2008).</li><li>Vintersten, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+in+red:+red+fluorescent+protein+expression+in+mouse+ES+cells,+embryos,+and+adult+animals.">Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals.</a> Genesis. 40 (4), 241-246 (2004).</li><li>Hayashi, S., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+recombination+in+diverse+tissue+by+a+tamoxifen-inducible+form+of+Cre:+a+tool+for+temporally+regulated+gene+activation/inactivation+in+the+mouse.">Efficient recombination in diverse tissue by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse.</a> Dev. Biol. 244 (2), 305-318 (2002).</li><li>Gong, S., Zheng, C., Doughty, M. L., Losos, K., Didkovsky, N., Schambra, U. B., Nowak, N. J., Joyner, A., Leblanc, G., Hatten, M. E., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+expression+atlas+of+the+central+nervous+system+based+on+bacterial+artificial+chromosomes.">A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.</a> Nature. 425 (6961), 917-925 (2003).</li><li>Mukouyama, Y. S., Deneen, B., Lukaszewicz, A., Novitch, B. G., Wichterle, H., Jessell, T. M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olig2++neuroepithelial+motoneuron+progenitors+are+not+multipotent+stem+cells+in+vivo.">Olig2+ neuroepithelial motoneuron progenitors are not multipotent stem cells in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 103 (5), 1551-1556 (2006).</li><li>Jones, E. A. V., Crotty, D., Kulesa, P. M., Waters, C. W., Baron, M. H., Fraser, S. E., Dickinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+in+vivo+imaging+of+postimplantation+mammalian+embryos+using+whole+embryo+culture.">Dynamic in vivo imaging of postimplantation mammalian embryos using whole embryo culture.</a> Genesis. 34, 228-235 (2002).</li><li>Piotrowska-Nitsche, K., Caspary, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+individual+cell+divisions+in+mouse+neuroepithelium+shows+asymmetry+in+cilium+formation+and+Sonic+hedgehog+response.">Live imaging of individual cell divisions in mouse neuroepithelium shows asymmetry in cilium formation and Sonic hedgehog response.</a> Cilia. 1 (6), (2012).</li><li>Nowotschin, S., Ferrer-Vaquer, A., Hadjantonakis, A. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mouse+development+with+confocal+time-lapse+microscopy.">Imaging mouse development with confocal time-lapse microscopy.</a> Methods in Enzymology. 476, 351-377 (2010).</li></ol>

利害の衝突は宣言されていない。

当社のex vivoでシステムが直接、リアルタイムで開発神経上皮内の単一細胞分裂を観察することを可能にします。例として、我々はマウス胚神経管の中に細胞分裂を検討し、繊毛形成またはShhの応答のいずれかを監視した。私たちは、イメージング結果（N = 24）私たちの技術は生理学的に適切なデータを提供示す固定部（N = 178）の結果と一致していたことを確認した。 ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Z/RED line (STOCK Tg(CAG-Bgeo,-DsRed*MST)1Nagy/J</td>
			<td>Jackson Laboratory</td>
			<td>005438</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olig2-eGFP line (STOCK Tg(Olig2-EGFP)EK23Gsat/Mmcd</td>
			<td>MMRRC,</td>
			<td>010555-UCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CAGGCreER</td>
			<td>Jackson Laboratory</td>
			<td>003724</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM/F12 (1:1)</td>
			<td>GIBCO</td>
			<td>21041-025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Newborn calf serum</td>
			<td>Lonza</td>
			<td>14-416F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rat Serum SD male</td>
			<td>Harlan Bioproducts</td>
			<td>4520</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1M Hepes</td>
			<td>BioWhittaker</td>
			<td>17-737E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>GIBCO</td>
			<td>21041-025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Light mineral oil</td>
			<td>Sigma</td>
			<td>M8410</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sstr3-GFP lentivirus</td>
			<td>Emory Viral Core</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro-knife, size 0.025 mm</td>
			<td>Electron Microscopy Sciences</td>
			<td>62091</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-L-lysine coated glass bottom dish</td>
			<td>MatTek</td>
			<td>P35GC-0-10-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100% petroleum jelly</td>
			<td>Kroger</td>
			<td>FL9958c</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A1R Laser Scanning Confocal Inverted Microscope</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NIS Elements software</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Imaris 3D software</td>
			<td>Bitplane AG</td>
			<td>Imaris 7.2.3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heat-inactivated sheep serum</td>
			<td>Invitrogen</td>
			<td>16210-072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Parafolmaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate Buffer</td>
			<td>Lab made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rat monoclonal anti-RFP (5F8)</td>
			<td>Chromotek</td>
			<td>110411</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit anti-Arl13b serum</td>
			<td>NeuroMab</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>mouse monoclonal anti-Arl13b 1:5</td>
			<td>NeuroMab</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit anti-Olig2</td>
			<td>Chemicon</td>
			<td>AB9610</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonals Pax6</td>
			<td>Developmental Hybridoma Bank</td>
			<td>Pax6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonalsShh</td>
			<td>Developmental Hybridoma Bank</td>
			<td>5E1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonals Nkx2.2</td>
			<td>Developmental Hybridoma Bank</td>
			<td>74.5A5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit polyclonal Ki67</td>
			<td>Abcam</td>
			<td>AB15580</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A11029</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568</td>
			<td>Molecular Probes</td>
			<td>A11031</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 350</td>
			<td>Molecular Probes</td>
			<td>A11046</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Fisher</td>
			<td>AC22989</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TO-PRO-3</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ProLong Gold anti-fade reagent</td>
			<td>Invitrogen</td>
			<td>P36934</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

ex vivo live imaging of single cell divisions in mouse neuroepithelium

私たちは、蛍光マーカーおよび胚マウス神経上皮の培養スライスのライブイメージングを統合するシステムを開発しました。私たちは、トレース系統遺伝細胞のための既存のマウス系統を利用しました：タモキシフェン誘導性CreのラインとのCreレポーターラインはCreを媒介組換えによりDsRedの発現。タモキシフェンの比較的低レベルを用いて、我々は我々は、個々の細胞分裂に従うことを可能にして、少数の細胞で組換えを誘導することができた。さらに、我々は、Olig2-EGFPトランスジェニックライン1-3を使用して、我々はウイルスが繊毛マーカー、SSTR3-GFP 4を発現培養スライスを感染させることにより繊毛の形成を監視シグナリング（シーッ）ソニック·ザ·ヘッジホッグに転写応答を観察した。イメージするためには神経上皮は、我々は神経管を単離し、E8.5で胚を採取し、撮影室への適切な培養条件の神経スライスをマウントし、タイムラプス共焦点イメージングを行った。当社EX生体ライブイメージング法は、一次繊毛形成と生理学的に適切な方法でShhの応答の相対的なタイミングを評価するために、単一の細胞分裂をトレースすることを可能にします。この方法は簡単明瞭な蛍光マーカーを使用して適応され、フィールドを、その場で 、リアルタイムで細胞の挙動を監視するためにどのとツールを提供しすることができます。

ここでは、E8.5マウス神経上皮内の単一細胞分裂のex vivoでのライブイメージングを行った。個々の細胞を標識するためには、組換え5,6（ 図3A）に基づいDsRedを表明したCreレポーター行を含む細胞のサブセットにCreリコンビナーゼを誘導した。したがって、48時間後に我々はex vivoでイメージング（ 図4A-D）の間に単一の細胞分裂を観察することがで?...

Watch this Scientific Journal Video about Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium at JoVE.com

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

ここでは、のために必要なツールを開発<em&gt; ex vivoで</emマウスE8.5神経上皮内の単一細胞分裂をトレースする&gt;ライブイメージング

ex vivoでマウス神経上皮における単一細胞分裂のライブイメージング

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

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Research

JoVE Journal

Neuroscience

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

10.3K ビュー.  Emory University School of Medicine. ここでは、のために必要なツールを開発 ex vivoでライブイメージング

ビデオ: Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

複数のマウスの神経解剖学的磁気共鳴イメージング

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

神経科学

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

開発神​​経上皮における細胞挙動の高解像度のライブイメージング

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

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 ...

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

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

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

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

共焦点肉眼検査を使用した生後小脳顆粒細胞の移動のex vivoイメージング

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

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同時<em&gt; ex vivoで</emによる二つの網膜の&gt;機能テスト<em&gt; in vivoで</em&gt;電図システム

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

単一セルの解像度蛍光は住む幼虫脳文化のショウジョウバエ概日時計のイメージング

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

Ex Vivoセル固有カルシウム マウス ダイヤフラムの三者のシナプスでシグナル伝達のイメージング

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

経瞳孔二光子マウス網膜のin vivoイメージング

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging in Mouse Inferior Olive

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

In vivo Calcium Imaging in Mouse Inferior Olive

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

インビボ マウス劣オリーブにおけるカルシウムイメージング

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

マウス Retina OCT ファイバーグラムと共焦点画像によるアライメント

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

著者スポットライト:緑内障の診断と治療を改善するためのIn vivoおよびex vivo網膜イメージングの進歩 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

可視光光干渉断層撮影ファイバーグラムと同一マウス網膜の共焦点像のアライメント

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...