著者らは、ヒマラヤスギシナイの再生医療研究所、ゲラン家族からサミュエルOschin総合がん研究所がん研究フォーラム賞からのサポートだけでなく、資金を承認したいと思います。記載されたプロジェクトは、研究資源、グラントUL1RR033176国立センターによって資金を供給CTSIコアバウチャーの形でサポートされ、トランスレーショナル科学、グラントUL1TR000124を進めるためのナショナルセンターで今した。内容はもっぱら著者の責任であり、必ずしもNIHの公式見解を示すものではありません。

この手順では、ヒマラヤスギシナイ動物実験委員会の要求に従う。調べでは、前に進むための制度IACUCのコンプライアンスを確保する必要があります。すべてのツールと​​試薬は使用前に滅菌する必要があります。 1。ツールの作成、ソリューション、DNA混合物<ol><li>マイクロピペットプラーに100 mmの火ポリッシュホウケイ酸ガラスキャピラリーチューブを挿入します。約17.5ミリメートルのピペットチップを形成するために、標準的な重み付きプルを可能にするために加熱を設定します。およそ直径100μmの開口部を作成するために、先端の先頭からの距離が約8〜9ミリメートルで鋭い外科ハサミで先端をカットします。 </li><li>ドライファストグリーンで（20μmのシリンジフィルターを利用する）フィルタリングヌクレアーゼフリー水を混合することにより、高速な緑色溶液w / vの1％株式を作る。 </li><li>トリス-EDTA（TE）緩衝液で希釈し、高度に濃縮されている精製されたエンドトキシンフリーのプラスミドDNA（ すなわち &gt; 3μgの/μl）を、分離します。プラスミドの所望の量を追加します。混合物に、TE緩衝液で希釈し（ファストグリーン0.1重量/容量％の最終濃度にする）、1％ファストグリーン溶液の1:10の比を加える。導入遺伝子の強い発現のためのプラスミドの0.5〜2.0μgの/μLの最終濃度を使用してください。 </li></ol> 2。動物麻酔、ピペットロード、軟膜表面プラスミドインジェクション<ol><li> 5-8分（PUP年齢/体重に依存時間）、氷上に置かれたペトリ皿に配置して1〜2日出生後のマウスに低体温麻酔を誘導する。低体温は、つま先つまむ、および/または尾レフからの移動の欠如によって確認されると、マウスが注入される準備ができている。痛みの反射が明らかである場合は、氷の上に戻って動物を配置します。低体温症、注射、エレクトロポレーション手順がそれに応じて適切な麻酔を維持するための時間に、注射手順とエレクトロポレーションは、合計未満2〜3分かかる。 </li><li>麻酔中に、標準的なピペットを使用して、慎重にピペットplasmiの量を所望D参照のためにパラフィンワックスフィルム片の上に（0.5-2μl）を注入される混ぜる。ここでは、プラスミドミックス0.5μLの全容量が注入される。次に、マイクロインジェクターを用いて、慎重にプラスミド混合物を含むチューブ内に組み立てられたガラスキャピラリーピペットを挿入する。それは、チューブの端に触れていないことを確認することにより、破損、先端を防ぐために、余分な予防措置をとる。ゆっくりと移動ダイヤルを背面ダイヤルすることによりピペットに溶液を吸引除去する。十分なボリュームがピペットにロードされると、0ヘクトパスカルの中立位置に圧力が戻るまで前方に回します。 </li><li> （2.2）からピペットパラフィンワックス膜基準噴射近くチップを配置しても軟膜表面/髄膜空間を充填するために適切な圧力を確保し、一方を排出するマイクロインジェクタからフットペダルを使用する注射用300〜450 hPaの圧力を用いてパラフィンワックス膜上にボリューム。イジェクト量は、以前にピペットでreferencに等しくなるまでそれに応じて圧力を調整Eのボリューム。 </li><li>先端が平衡化されており、麻酔が確認された後、仔を注入する準備ができている。 </li><li>実験の目的は、軟膜表面前駆細胞（ 図1A）内に下方DNAのエレクトロポレーションを可能にするために頭蓋下及び軟膜表面の上方プラスミド混合物を注入することである。皮質注射のために、あまり利き手の親指と人差し指を使って子犬を押したままにします。皮質半球など上丘など他の表面的な構造は、これらの構造（ 図1B）の容易なターゲティングを可能にP0とP2との間表示されます。皮質の利き手とターゲット所望の領域（等すなわちモーター、体性感覚）を用いて、脳動脈を避けるように注意しながら、皮膚や頭蓋骨を過ぎピペットを挿入します。 （ちょうど頭蓋骨を貫通した後に、さらに抵抗性の欠如によって示されます）頭蓋骨過去先端のさらなる浸透を防ぐようにしてください。 </li><li>血漿を注入IDソリューションマイクロインジェクタのフットペダルを使用して、組織（ 図2A）に均等に分散させることを確認してください。 注意：これは、経験的に、流体圧力による血腫を回避しながら、プラスミド送達を最大化するアプローチを決定することは非常に重要である。これは、注入の持続時間、角度、体積、および血管破壊を回避するために正確な標的部位を調整することによって行うことができる。 </li><li>試してみて、パラフィン紙またはプラスチックパラフィンフィルム上に以前に行った注入からの試験液滴でチップを配置することによって、注射の間に乾燥するの先端を保つようにしてください。これは、チップと矛盾送達体積の目詰まりをもたらすことができる蒸発させ、結晶化DNA溶液の先端の蓄積を防止する必要がある。 </li></ol> 3。エレクトロ<ol><li>各パルス間に950ミリ秒間隔で、50ミリ秒続く、5パルスの135から150 Vのエレクトロポを設定します。コンダクタンスを増加させるために、及び皮膚の燃焼を防止するため、電気穿孔ゲルで3 mmのプラチナtweezertrodesをカバーしています。麻酔を確実にするために、この時点でつま先つまんで尾反射神経をチェックしてください。応答性の場合、麻酔を数分間氷上に置きます。 </li><li>皮質（および上丘）注射のために、（ -脳室帯のエレクトロポレーションに使用され、10mmの電極7と比較して）仔の生存率を増加させるために3mmの電極（ 図2B）を使用する。注入された領域の上に電極と電極の向きが正中線（ 図2C）に45〜60°の角度であるように、目下の反対側の領域の周囲正に帯電したプローブの負に帯電したプローブを配置します。 </li><li>曲率を占めているときの電流をトリガーするエレクトロのフットペダルを使用して、効果的に基礎と軟膜表面の細胞にDNAを対象に、PUPの年齢と体重に応じて、3〜5パルスのための場所でtweezertrodesを開催半球の。 注：負極のパルスまたは大きな電極がエレクトロポ面積を増加させるために使用することができる間に注入領域にわたって掃引することができる。 </li><li>すぐにエレクトロポレーション後、慎重に子犬からゲルをきれいし、約5分間加熱ランプの下に配置します。 </li><li> PUPは急性チアノーゼによるエレクトロポレーション後数分で、自然の赤みがかったピンクの色合いを失うことになる。それらのケージに戻す前に、自然な色と通常の移動の開始を回収するために子犬を観察します。 </li><li>彼女が戻って巣に子犬をもたらし、それらをグルーミング始まるかどうか観察することによって、母親と再社会化を確実にする。彼女は攻撃的になった場合、必ず子犬がゲルの残党があり、子犬に臭いを転送するための寝具のいくつかと、それらをインキュベートしないようにしてください。 </li></ol>上丘を標的とする4。変更 ターゲティング： <ol><li>麟蹄皮質と同じ方法で上丘CTが、対象領域がちょうど後方ラムダへとおよそ正中線（ 図3Aと図3B）の横にあることに注意してください。成功した注射で、プラスミドミックスが自然に上丘の輪郭を塗りつぶします。注射は正常に行われると、動物は、エレクトロポレーションの準備ができている。 </li></ol> エレクトロポレーション： <ol><li>非常によく似た方法で、上丘の注射のために、上丘の表面的な領域（ 図3C）にDNAを駆動し、注入されたエリアと目、鼻やあごの周りに正に帯電したプローブ上には、電極の負に帯電したプローブを配置。この場合、電極の向きは正中線に対して25〜40°の角度にある。 </li></ol>

<ol>
	<li>Breunig, J. J., Haydar, T. F., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells:+historical+perspective+and+future+prospects.">Neural stem cells: historical perspective and future prospects.</a> Neuron. 70 (4), 614-625 (2011).</li><li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+neural+stem+cells.">Mammalian neural stem cells.</a> Science. 287 (5457), (2000).</li><li>Kriegstein, A., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+nature+of+embryonic+and+adult+neural+stem+cells.">The glial nature of embryonic and adult neural stem cells.</a> Annu Rev Neurosci. 32, 149-184 (2009).</li><li>Breunig, J. J., Arellano, J. I., Macklis, J. D., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everything+that+glitters+isn't+gold:+a+critical+review+of+postnatal+neural+precursor+analyses.">Everything that glitters isn't gold: a critical review of postnatal neural precursor analyses.</a> Cell Stem Cell. 1 (6), 612-627 (2007).</li><li>Costa, M. R., Kessaris, N., Richardson, W. D., Gotz, M., Hedin-Pereira, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginal+zone/layer+I+as+a+novel+niche+for+neurogenesis+and+gliogenesis+in+developing+cerebral+cortex.">The marginal zone/layer I as a novel niche for neurogenesis and gliogenesis in developing cerebral cortex.</a> J Neurosci. 27 (42), 11376-11388 (2007).</li><li>Breunig, J. J., 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=Rapid+genetic+targeting+of+pial+surface+neural+progenitors+and+immature+neurons+by+neonatal+electroporation.">Rapid genetic targeting of pial surface neural progenitors and immature neurons by neonatal electroporation.</a> Neural Dev. 7, (2012).</li><li>Ohira, K., 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=Ischemia-induced+neurogenesis+of+neocortical+layer+1+progenitor+cells.">Ischemia-induced neurogenesis of neocortical layer 1 progenitor cells.</a> Nat Neurosci. 13 (2), 173-179 (2010).</li><li>Bystron, I., Blakemore, C., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+human+cerebral+cortex:+Boulder+Committee+revisited.">Development of the human cerebral cortex: Boulder Committee revisited.</a> Nat Rev Neurosci. 9 (2), 110-122 (2008).</li><li>Schmechel, D. E., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Golgi+study+of+radial+glial+cells+in+developing+monkey+telencephalon:+morphogenesis+and+transformation+into+astrocytes.">A Golgi study of radial glial cells in developing monkey telencephalon: morphogenesis and transformation into astrocytes.</a> Anat Embryol (Berl. 156 (2), 115-152 (1979).</li><li>Halfter, W., Dong, S., Yip, Y. P., Willem, M., Mayer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+function+of+the+pial+basement+membrane+in+cortical+histogenesis.">A critical function of the pial basement membrane in cortical histogenesis.</a> J Neurosci. 22 (14), 6029-6040 (2002).</li><li>Bystron, I., Rakic, P., Molnar, Z., Blakemore, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+neurons+of+the+human+cerebral+cortex.">The first neurons of the human cerebral cortex.</a> Nat Neurosci. 9 (7), 880-886 (2006).</li><li>Tanaka, D. H., Maekawa, K., Yanagawa, Y., Obata, K., Murakami, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidirectional+and+multizonal+tangential+migration+of+GABAergic+interneurons+in+the+developing+cerebral+cortex.">Multidirectional and multizonal tangential migration of GABAergic interneurons in the developing cerebral cortex.</a> Development. 133 (11), 2167-2176 (2006).</li><li>Ang Jr, E. S., Haydar, T. F., Gluncic, V., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-dimensional+migratory+coordinates+of+GABAergic+interneurons+in+the+developing+mouse+cortex.">Four-dimensional migratory coordinates of GABAergic interneurons in the developing mouse cortex.</a> J Neurosci. 23 (13), 5805-5815 (2003).</li><li>Tamamaki, N., Fujimori, K. E., Takauji, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+route+of+tangentially+migrating+neurons+in+the+developing+neocortical+intermediate+zone.">Origin and route of tangentially migrating neurons in the developing neocortical intermediate zone.</a> J Neurosci. 17 (21), 8313-8323 (1997).</li><li>Larkum, 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=The+yin+and+yang+of+cortical+layer+1.">The yin and yang of cortical layer 1.</a> Nat Neurosci. 16 (2), 114-115 (2013).</li><li>Jiang, X., Wang, G., Lee, A. J., Stornetta, R. L., Zhu, J. 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+organization+of+two+new+cortical+interneuronal+circuits.">The organization of two new cortical interneuronal circuits.</a> Nat Neurosci. 16 (2), 210-218 (2013).</li><li>Endo, T., Isa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+different+AMPA-type+glutamate+receptors+in+morphologically+identified+neurons+in+rat+superficial+superior+colliculus.">Functionally different AMPA-type glutamate receptors in morphologically identified neurons in rat superficial superior colliculus.</a> Neuroscience. 108 (1), 129-141 (2001).</li><li>Schmidt, M., Ozen Boller, M., G, W. C., Hall, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disinhibition+in+rat+superior+colliculus+mediated+by+GABAc+receptors.">Disinhibition in rat superior colliculus mediated by GABAc receptors.</a> J Neurosci. 21 (2), 691-699 (2001).</li><li>Ackman, J. B., Burbridge, T. J., Crair, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+waves+coordinate+patterned+activity+throughout+the+developing+visual+system.">Retinal waves coordinate patterned activity throughout the developing visual system.</a> Nature. 490 (7419), 219-225 (2012).</li><li>De Marco Garcia, N. V., Karayannis, T., 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=Neuronal+activity+is+required+for+the+development+of+specific+cortical+interneuron+subtypes.">Neuronal activity is required for the development of specific cortical interneuron subtypes.</a> Nature. 472 (7343), 351-355 (2011).</li><li>Boller, M., Schmidt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+maturation+of+GABA(A)+and+GABA(C)+receptor+function+in+the+mammalian+superior+colliculus.">Postnatal maturation of GABA(A) and GABA(C) receptor function in the mammalian superior colliculus.</a> Eur J Neurosci. 14 (8), 1185-1193 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev Biol. 240 (1), 237-246 (2001).</li><li>De Vry, J., 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=In+vivo+electroporation+of+the+central+nervous+system:+a+non-viral+approach+for+targeted+gene+delivery.">In vivo electroporation of the central nervous system: a non-viral approach for targeted gene delivery.</a> Prog Neurobiol. 92 (3), 227-244 (2010).</li><li>Stancik, E. K., Navarro-Quiroga, I., Sellke, R., Haydar, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+ventricular+zone+neural+precursors+contributes+to+neuronal+fate+diversity+in+the+postnatal+neocortex.">Heterogeneity in ventricular zone neural precursors contributes to neuronal fate diversity in the postnatal neocortex.</a> J Neurosci. 30 (20), 7028-7036 (2010).</li><li>De la Rossa, A., 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=In+vivo+reprogramming+of+circuit+connectivity+in+postmitotic+neocortical+neurons.">In vivo reprogramming of circuit connectivity in postmitotic neocortical neurons.</a> Nat Neurosci. 16 (2), 193-200 (2013).</li><li>Boutin, C., Diestel, S., Desoeuvre, A., Tiveron, M. C., Cremer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vivo+electroporation+of+the+postnatal+rodent+forebrain.">Efficient in vivo electroporation of the postnatal rodent forebrain.</a> PLoS One. 3 (4), (2008).</li><li>Chesler, A. T., Le Pichon, C. E., Brann, J. H., Araneda, R. C., Zou, D. J., Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+gene+expression+by+postnatal+electroporation+during+olfactory+interneuron+nurogenesis.">Selective gene expression by postnatal electroporation during olfactory interneuron nurogenesis.</a> PLoS One. 3 (1), (2008).</li><li>dal Maschio, 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=High-performance+and+site-directed+in+utero+electroporation+by+a+triple-electrode+probe.">High-performance and site-directed in utero electroporation by a triple-electrode probe.</a> Nat Commun. 3, (2012).</li><li>Yoshida, A., Yamaguchi, Y., Nonomura, K., Kawakami, K., Takahashi, Y., Miura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+expression+of+different+transgenes+in+neurons+and+glia+by+combining+in+utero+electroporation+with+the+Tol2+transposon-mediated+gene+transfer+system.">Simultaneous expression of different transgenes in neurons and glia by combining in utero electroporation with the Tol2 transposon-mediated gene transfer system.</a> Genes Cells. 15 (5), 501-512 (2010).</li><li>Chen, F., LoTurco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+stable+transgenesis+of+radial+glia+lineage+in+rat+neocortex+by+piggyBac+mediated+transposition.">A method for stable transgenesis of radial glia lineage in rat neocortex by piggyBac mediated transposition.</a> J Neurosci Methods. 207 (2), 172-180 (2012).</li><li>Feliciano, D. M., Lafourcade, C. A., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+subventricular+zone+electroporation.">Neonatal subventricular zone electroporation.</a> J Vis Exp. , (2013).</li><li>Lam, A. J., 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=Improving+FRET+dynamic+range+with+bright+green+and+red+fluorescent+proteins.">Improving FRET dynamic range with bright green and red fluorescent proteins.</a> Nat Methods. 9 (10), 1005-1012 (2012).</li><li>Subach, O. M., Cranfill, P. J., Davidson, M. W., Verkhusha, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enhanced+monomeric+blue+fluorescent+protein+with+the+high+chemical+stability+of+the+chromophore.">An enhanced monomeric blue fluorescent protein with the high chemical stability of the chromophore.</a> PLoS One. 6 (12), (2011).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

軟膜表面前駆細胞の正常なエレクトロポレーションのための最も重要な側面は、次のとおりです。1）軟膜表面にプラスミドミックスの標的; 2）注射部位における血腫の発生を回避すること;および3）中脳電気穿孔に関連した死亡率を回避する。 適切に標的化は、軟膜表面は軟膜表面の浸透を避けるために、頭蓋骨の測定と慎重なパンクチャリングすることによって達成?...

神経幹細胞および前駆細胞は、哺乳動物CNS 1、2を通して存在している。その性質や脳と脊髄の心室領域を取り巻く胚および成体胚ゾーンの特性は、広範囲過去十年間1-3に記載されています。大部分は、これは、このようなフロックス対立遺伝子または4をトレースするレトロウイルス系統の神経系の特定のCre組換えとしてますます正確な遺伝的なツールの開発になっている。しかし、1前駆地域軟膜表面前駆ゾーンは、しているのはごく最近では詳細5-7に説明され、総合的な検討を待ってさ。 脳の軟膜表面は、脳の表面と周囲の髄膜8との間のインタフェースとして定義される。後の開発、神経上皮と、中は、放射状グリアエンドフィートは、この表面9,10に接続します。モミの一部人間の脳およびニューロンの多くの有糸分裂における番目のニューロンは、この領域11において観察される。その後、胚の神経発生の間に、皮質ニューロンは、中間ゾーンおよび脳室下帯12-14におけるそれらの遊走の経路に加えて、軟膜領域を横断することが知られている。この期間中に、幹細胞は、このゾーンから培養することができる、それは神経およびグリア5の活性部位であると思われる。成体の脳において、介在ニューロンは、低酸素チャレンジ7以下の軟膜表面前駆細胞から生まれることができることが報告されている。しかし、胚および生後発育中にgenensisに彼に、この領域の寄与は、特にこの領域6を調査することの困難に一部あいまいなままである。上丘におよび大脳皮質において、表面的な（または皮質の層I）介在ニューロンは、下にある興奮性ニューロン集団の回路出力を変調するため、significを貢献するかもしれないこれらの構造の機能にantly。具体的には、レイヤ1介在ニューロンは、皮質の列15,16の表層と深層に彼らの広範な接続性与えられた大脳皮質の上位層を通してニューロンの発火を調整するために主要な位置にある。同様に、水平ニューロンは、比較的広い面積にわたって皮質および網膜繊維、プロジェクトから興奮性入力を受信し、遠隔視覚刺激17,18に応答ニューロン集団の阻害を媒介すると推測される。また、その形態は現像視覚系19におけるパターン化された波の活動に潜在的な役割を果たしてよく適している。興味深いことに、ニューロンの発達と成熟は、出生後大幅に起こります。また、この成熟過程は、神経活動によって調節されることが見出されているので、回路機能20,21上の生涯結果を伴う発達可塑性の基板である。注目すべきことに、NOプロモーターは、特にトランスジェニックこれらの細胞を標的とすることができるが記載されている。除する前駆細胞は、レトロウイルス7でターゲティングすることができるが、ウイルス産生は時間がかかり、細胞形質導入のために必要な高い力価を得るために熟練を要する。 それが神経前駆細胞4、22、23におけるシグナル伝達経路の迅速かつ効率的な遺伝的尋問を可能にするので、エレクトロポレーションは、神経発達の研究でルネサンスにつながっている。エレクトロポレーションは、一方向4脳室の周囲の増殖性前駆細胞にDNAを駆動するために、ヘッドの外部への電気パルスの送出に続いて、プラスミドDNAの注入を伴う22、23。エレクトロポレーションは、プラスミドの導入遺伝子24の発現のために細胞周期のM期を通過する細胞の通過を必要とするように見える。具体的には、それだけであることが見出されているプラスミドのエレクトロポレーションの8時間以内にM期を通過する細胞は、心室壁24の〜160ミクロン内の全ての細胞への効果的な配信にもかかわらず、導入遺伝子を発現する。これは、核透過性を引き起こす化学物質が有糸分裂後の細胞25内のプラスミドの発現を誘導することができるように、これは、エピソームプラスミドの核へのアクセスを可能に核膜破壊の必要性によるものであると推測される。もともと胚22で用い、エレクトロポレーションは、ずっと後26,27生後脳内での使用に適合した。最近では、軟膜表面前駆6の遺伝子操作で使用するためのエレクトロポレーションを適応している。また、このアプローチを使用して我々は、前駆体の二つの異なる系統がこの領域-介在ニューロンおよびアストロサイト6に明らかに存在することが示されている。このプロトコルは、尋問のためにこれらの細胞を標的とするために簡単、迅速、かつ強力な方法を詳述これらの細胞の発達を調節する機構の。

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			<td height="21" style="height:21px;width:248px;">Item Name</td>
			<td style="width:269px;">Vendor</td>
			<td style="width:177px;">Catalog Number</td>
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			<td height="21" style="height:21px;">Fire Polished Borosilicate Tubing</td>
			<td>World Precision Instruments, Inc.</td>
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			<td height="21" style="height:21px;">Micropipette Puller</td>
			<td>Sutter Instruments Company</td>
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			<td>Sutter Instruments Company</td>
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			<td><a name="RANGE!B6">Harvard Apparatus, BTX Instrument Div</a></td>
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			<td height="21" style="height:21px;">3mm Platinum Tweezertrodes</td>
			<td>Harvard Apparatus, BTX Instrument Div</td>
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			<td>Cardinal Health</td>
			<td>70315-025</td>
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			<td height="22" style="height:22px;">Tris-EDTA Buffer, pH 8.0</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>11-01-02-05</td>
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			<td height="21" style="height:21px;">Infrared Heat Lamp</td>
			<td>VWR</td>
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neonatal pial surface electroporation

過去数年間で軟膜表面は損傷後を含め、胚周産期および成人の神経およびグリア発生時の重要性の胚ニッチとして同定されている。しかしながら、これらの遺伝的に前駆細胞集団に問い合わせ、それらの系統を追跡するための方法は、特異性またはウイルスの産生時間を消費がないため限定されていた。したがって、この領域での進歩はこの場所の調査のほんの一握りでは比較的遅れている。エレクトロポレーションは、胚の神経幹細胞の特性を研究し、より最近では生後脳におけるする十年間使用されている。ここでは、適合したエレクトロポレーション法に基づく軟膜表面前駆細胞の遺伝子操作のための、効率的で迅速な、かつ簡単な手法について説明します。軟膜表面エレクトロポレーションは、このようにこれらの細胞を研究するための時間の節約で経済的なアプローチを表す、これらの前駆細胞の容易な遺伝標識及び操作を可能にする。

細胞-主に前駆細胞-AT軟膜表面6またはそれに近い中でのプラスミドDNAの発現の軟膜表面のエレクトロポレーションの結果。具体的には、電極の向きは、プラスミドの動きとその後の発現の方向を口述において重要である。従って、二重電極構成において、プラスミドは、負と正電極との間のほぼ直線状ベクターに向けられる。負極が注射部位の上に配置し、正極、負極に腹ですしてお...

Watch this Scientific Journal Video about Neonatal Pial Surface Electroporation at JoVE.com

Neonatal Pial Surface Electroporation

軟膜表面はますます注目を受けているCNSにおけるユニークな前駆ゾーンです。ここで、我々の詳細を修飾エレクトロポレーション法を使用して、この前駆ゾーンの迅速な遺伝子操作のための方法。この手順は、細胞系統および細胞分化に関与するシグナル伝達経路の細胞および分子研究のために使用することができ、娘細胞の運命および特性を解明する。

新生児軟膜表面エレクトロ

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Research

JoVE Journal

Neuroscience

13.8K ビュー.  Cedars-Sinai Medical Center. 軟膜表面はますます注目を受けているCNSにおけるユニークな前駆ゾーンです。ここで、我々の詳細を修飾エレクトロポレーション法を使用して、この前駆ゾーンの迅速な遺伝子操作のための方法。この手順は、細胞系統および細胞分化に関与するシグナル伝達経路の細胞および分子研究のために使用することができ、娘細胞の運命および特性を解明する?...

ビデオ: Neonatal Pial Surface Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

神経科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

開発マウス網膜の in vivoエレクトロポレーションで

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

マウス子宮内でエレクトロポレーション：制御時空間遺伝子導入

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

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 ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

皮質介在ニューロンのサブタイプ選択的エレクトロポレーション

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

新しい方法を用いた自由に動く新生児ラットの脳波記録

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

新生児マウスからの脊髄ニューロンの単離と培養

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

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, ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

生体内で新生仔マウスの大脳皮質ニューロンの 2 光子イメージング

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

大人のマウスにおける人工内耳表面製剤

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...