この作品は、神経疾患や脳卒中の助成金（CMT）にNS081333の国立研究所によってサポートされていました、アルツハイマー病協会の助成金NIRG-10から171721と国立精神衛生研究所（UHに）MH096702を付与し、高齢化資金によるアルツハイマー病研究に関する国立研究所（YYJとJBに）コロンビア大学パイロットグラントAG008702でセンター。

注：すべての動物実験では、実験動物の管理と使用のための制度や国のガイドラインに従いました。 1.外科手術器具およびソリューションの準備<ol><li>すべてのステンレス鋼の外科手術用器具をオートクレーブ。 </li><li>無菌の分子等級脱イオン蒸留水で200プルーフ無水エタノールを希釈することにより、70％エタノールを調製します。 </li><li>ハミルトンシリンジに29 G針を取り付けます。汲み上げ、1分間繰り返しナノ純水を噴射してハミルトン注射器と針の内部を清掃してください。 70％エタノールで、この手順を繰り返します。 <ol><li>ハミルトンシリンジからプランジャーと針を外します。空気は、層流フードO / Nで部品を乾燥させます。 </li><li>使用前に、30分間紫外線を針及び注射器を照射します。 </li></ol></li><li> 0.9％の体積濃度を最終重量に分子グレードの水に塩化ナトリウムを溶解して生理食塩水を調製します。 Sterili0.2μmの細孔のフィルターを通してそれをフィルタリングすることによってソリューションをぜ。 </li></ol> 2.オリゴマーAβ1-42の準備<ol><li> FA」など13の出版物に記載されているように単量体化とは、正確に5 mMのDMSO中で組換えヒトAβ1-42を再懸濁します。 </li><li>手術前日に100μMの滅菌（1×）、PBSで5 mMのAβ1-42を希釈します。 </li><li>粉砕してよく、溶液を混ぜます。 </li><li> 4℃で12時間、Aβ1-42溶液をインキュベートします。 注：このようなPBS中にDMSOを希釈またはスクランブルなどのコントロール·ソリューションは、/Aβ1-42ペプチドは、Aβ1-42と同じように準備する必要があります逆にします。 </li></ol> 3.注入座標を決定<ol><li>正確な前後（AP）、内側-外側（ML）を決定するために、成体マウスの脳アトラスを使用し、背腹（DV）は、関心14の脳領域の座標。 いいえTE：私たちはブレグマから次の座標で対象と海馬の歯状回を選んだ技術説明するために：AP、-2.00ミリメートル。 MLは、1.3ミリメートルを±します。 DV、-2.2 mmです。 AP値の前に負の符号は、ブレグマの2.00ミリメートルの後方であることを示しています。 ML値を先行±符号は、中心から左右の方向を示しています。最後に、DVの前に負の符号は、それが脳の表面から腹側に移動していることを示します。 </li></ol> 4.定位フレームの設定<ol><li>外科用フェイスマスク、きれいな白衣、および無菌手術用手袋を着用してください。 </li><li>定位固定装置、70％エタノールでマウスアダプタを拭います。 </li><li>カウンターの上に滅菌外科用ドレープを下にして置きます。 </li><li>滅菌外科用ドレープの上にマウス·アダプタとの定位固定装置を配置します。 </li><li> 70％エタノールでマウスの加熱パッドを拭きます。定位フレームのベッドの上に加熱パッドを配置します。汎用laboraを使用しますマウスアダプタベッドの上に加熱パッドダウンテープに保守党表示用テープ。 <ol><li>製造業者の指示に従って温水再循環ポンプに加熱パッドを取り付けます。 </li><li>温水再循環ポンプの電源を入れ、37℃に温度を設定し、それが温度に達するまで待ちます。 </li></ol></li><li>製造業者の指示に従って、電動垂直注入軸にそれをねじ込むことにより、定位フレーム上に29 G針で50μlのハミルトンシリンジを貼り付けます。 </li><li>ビーズ滅菌器の電源を入れ、メーカーの設定温度に達するまで待ちます。 注：これは、動物間のステンレス鋼器具を滅菌するために使用されます。これは、楽器を殺菌するためのビーズとの接触の20秒かかります。 </li><li>ホットプレートの電源を入れ、42℃に設定し、上にきれいな空のケージを置きます。 </li><li>ハミルトンシリンジを定位フレームに固定されているが、電動定位のIを使用njectorAβ1-42溶液を描画します。 注意：シリンジに溶液を4つ以上μLを描画し、その後、注入量を設定するために定位注射器を使用しています。製造元の指示に係る注射器を操作してください。 </li></ol> 5.動物の準備<ol><li>重量計を用いてマウスの重量を決定します。 </li><li>腹腔内（IP）を注入することにより、100 mgの/ kgのケタミンおよび10mg / kgのキシラジンでケタミン/キシラジンのカクテルで、マウスを麻酔。つま先ピンチ反射の消失によって麻酔深度を監視します。 </li><li>マウスが鎮静された後にバリカンを取ると頭蓋骨上の皮膚を露出させるためにその頭を剃ります。 </li><li>定位ベッドの上に加熱パッドの上にマウスを置きます。 </li><li>口を開いて、歯のガードの内側切歯を配置するために、スパチュラを使用します。マウスの顔にノーズピースを固定します。静かに、あまりにもタイトではない、それを取り締まります。 </li><li>ポジ頭を保護するために外耳道に二国間の耳のクロスバーをTION。それは頭蓋骨に当たるまでの各クロスバーを移動し、それをロックするネジを回します。 <ol><li>頭がそっと頭を下にプッシュする人差し指を使用し固定されていることを確認します。ヘッドが適切に固定されている場合はそれが出て与えることはありませんか押すと移動します。 </li></ol></li><li>湿ったそれらを保つために、目にアイクリームまたは点眼の低下を適用します。目は、外科的処置を通して湿っていることを確認してください。 </li><li>手術部位を消毒するには、70％エタノール、続いて頭蓋骨を覆う皮膚にベータダイン液を、適用するために滅菌綿棒を使用しています。 2回以上清掃交流ベタジンおよび70％エタノールを実行します。 </li></ol> 6.手術と輸液<ol><li>頭皮の正中線2-3ミリの切開を作るためにメスを使用してから、頭蓋骨、ランドマークの矢状縫合、ブレグマとラムダを露出させるために1.0〜1.5センチメートルに切開線を拡張するためにまっすぐ細かいハサミを使用します定位座標が基づいているの。離れて肌を保つために、マイクロクランプを使用してください。 注：ブレグマとラムダ位置はマウスの脳アトラス」定位座標でマウス脳&quot;14で説明されています。 </li><li> 、綿棒を取り、滅菌生理食塩水でそれを浸して、頭蓋骨をきれいにするためにそれを使用。その後、頭蓋骨を乾燥させるためにきれいな乾いた綿棒を使用しています。 </li><li>ブレグマにドットをマークするために、滅菌細かい点のペンを使用してください。針の先端がちょうどブレグマ上のドットに接触するようにハミルトンシリンジを配置するために、定位マイクロマニピュレーターを使用してください。 <ol><li>座標DVを記録します。 </li><li>針の先端は、ラムダ点に接触するように頭蓋骨のAP軸がレベルであるかどうかを確認するには、後方にハミルトンシリンジを移動します。 </li><li> DVの位置を記録します。 0.5ミリメートルの範囲内であるDVはブレグマとラムダの座標を確認してください。 注：目標は、ブレグマとラムダの間のDV差を最小にすることです。 </li></ol></li><li> mを使用しますicromanipulatorはブレグマドットに戻るハミルトン注射針の先端を返します。 </li><li>開始APの位置を記録します。 </li><li>出発APから2.00ミリメートルの座標を減算して終了APの位置を計算します。 </li><li>座標最終APにハミルトンシリンジの針の位置を変更するためにマイクロマニピュレーターを使用してください。 </li><li>現在のML座標記録します。 </li><li>左側を計算し、右MLを終了すると、座標ML開始から1.3ミリメートルを加算または減算することによって調整します。 </li><li>終了ML座標のいずれかにハミルトンシリンジ針を移動させるためにマイクロマニピュレーターを使用してください。 <ol><li>両方の終了ML座標上の頭蓋骨の表面に針先をタッチし、DVの座標を記録し、値が0.5ミリメートルの範囲内であることを確認してください。 注：目標は、2つの終了ML座標間のDV差を最小にすることです。 </li><li>終了時に頭蓋骨の上に0.5〜1センチメートルまで針を引っ張る座標間。目にドットをマークするために、滅菌細かい点ペンを取ります頭蓋骨のE面。頭蓋骨の反対側にこの手順を繰り返します。 </li></ol></li><li>邪魔にならないように明確なハミルトンシリンジをスイング。 </li><li>ドリルに0.8ミリメートルのドリルヘッドを取り付けます。 、両手でドリルを取る安定性のためにテーブルの表面上に肘を設定し、わずかに左または右ML座標のいずれかでシャーピードットの上にドリルヘッドを配置します。ドリルをアクティブにし、頭蓋骨に穴を導入するために頭蓋骨の表面にドリル先端を下げます。反対側に、この手順を繰り返します。 注：一部の出血が新たに導入された孔から発生することがあります。そこ出血している場合は、血液を軽くするため、クリーン乾いた綿棒を使用しています。 </li><li>新たに導入されたML穴のいずれかを介して元の位置に戻しハミルトンシリンジを移動します。ちょうど頭蓋骨を過ぎハミルトン針を下げます。この時点で脳を穿刺しないように注意してください。確認するために針がシュール頭蓋骨が人差し指を取り、静かにするために頭蓋骨に対して針を押して通過しました電子針は穴を終了しません。 </li><li>出発DV座標を記録します。 </li><li>終了DVが開始座標DVから2.2ミリメートルを減算することにより座標を計算します。 </li><li>座標ダウン終了DVに針を下ろします。 </li><li> 0.5μL/分の速度で歯状回にAβ1-42の4μLをポンピングする定位噴射ポンプをアクティブにします。 </li><li>注入が完了すると、針が注入部位から溶液の逆流を最小限に抑えるために追加の1分間の場所に留まるう。 </li><li>脳の反対側に針を移動し、ステップ6.13から6.18を繰り返します。 </li></ol> 7.動物の取り外しおよび術後ケア<ol><li>耳のバーや顔/鼻ガードを外します。装置からマウスを外します。 </li><li>頭皮を閉じて、縫合糸で傷をシールプルする学生標準パターン鉗子を使用してください。 </li><li>水分補給のためのIPを介してマウスに滅菌生理食塩水の1ミリリットルを注入します。 </li><li>で痛みを緩和するジェクトブプレノルフィン（0.1 mgの/ kg）を皮下に投与することができます。 </li><li>それが起きるまで、42℃のホットプレートの上に設定し、クリーン空のケージにマウスを維持し、十分な食料と水をそのハウジングケージに戻します。 </li><li>皮下注射を介して痛みの軽減のために3日間の12時間毎にブプレノルフィンを投与します。 </li></ol> 8.抜糸<ol><li> IPによっては100mg / kgのケタミンおよび10mg / kgのxlyazineでケタミン/キシラジンのカクテルで、マウスを麻酔。注：これは、7〜10日、手術後に行われます。 </li><li>縫合糸を除去するために、学生の標準パターン鉗子とストレート細かいハサミを使用してください。 </li><li>水分補給のためのIPを介してマウスに滅菌生理食塩水の1ミリリットルを注入します。 </li><li>それが起きるまで、42℃のホットプレートの上にきれいな空のケージセットでマウスを維持し、その後、十分な食料と水をそのハウジングケージに戻します。 </li></ol> 9.動物の犠牲と組織処理<ol><li>マウスとpを麻酔、4％パラホルムアルデヒド15でそれをerfuse脳を除去し、16を凍結切片のためにそれを処理します。 注：動物の犠牲のタイミングは、実験者に依存します。しかし、私たちの研究のために、私たちは7日及び14日に手術を投稿しました。 </li></ol>

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	<li>Baleriola, 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=Axonally+Synthesized+ATF4+Transmits+a+Neurodegenerative+Signal+across+Brain+Regions.">Axonally Synthesized ATF4 Transmits a Neurodegenerative Signal across Brain Regions.</a> Cell. 158 (5), 1159-1172 (2014).</li><li>Haass, C., Selkoe, 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=Soluble+protein+oligomers+in+neurodegeneration:+lessons+from+the+Alzheimer's+amyloid+beta-peptide.+Nature+reviews.">Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nature reviews.</a> Molecular cell biology. 8 (2), 101-112 (2007).</li><li>Knowles, J. 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=The+p75+neurotrophin+receptor+promotes+amyloid-beta(1-42)-induced+neuritic+dystrophy+in+vitro+and+in+vivo.">The p75 neurotrophin receptor promotes amyloid-beta(1-42)-induced neuritic dystrophy in vitro and in vivo.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 29 (34), 10627-10637 (2009).</li><li>Troy, C. 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=beta-Amyloid-induced+neuronal+apoptosis+requires+c-Jun+N-terminal+kinase+activation.">beta-Amyloid-induced neuronal apoptosis requires c-Jun N-terminal kinase activation.</a> Journal of neurochemistry. 77 (1), 157-164 (2001).</li><li>Crews, L., Rockenstein, E., Masliah, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APP+transgenic+modeling+of+Alzheimer's+disease:+mechanisms+of+neurodegeneration+and+aberrant+neurogenesis.">APP transgenic modeling of Alzheimer's disease: mechanisms of neurodegeneration and aberrant neurogenesis.</a> Brain structure &amp; function. 214 (2-3), 111-126 (2010).</li><li>Gotz, J., Ittner, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+Alzheimer's+disease+and+frontotemporal+dementia.">Animal models of Alzheimer's disease and frontotemporal dementia.</a> Nature reviews. Neuroscience. 9 (7), 532-544 (2008).</li><li>Calhoun, M. 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=Neuron+loss+in+APP+transgenic+mice.">Neuron loss in APP transgenic mice.</a> Nature. 395 (6704), 755-756 (1998).</li><li>Oakley, H., 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=Intraneuronal+beta-amyloid+aggregates,+neurodegeneration,+and+neuron+loss+in+transgenic+mice+with+five+familial+Alzheimer's+disease+mutations:+potential+factors+in+amyloid+plaque+formation.">Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 26 (40), 10129-10140 (2006).</li><li>Jean, Y. Y., 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=Caspase-2+is+essential+for+c-Jun+transcriptional+activation+and+Bim+induction+in+neuron+death.">Caspase-2 is essential for c-Jun transcriptional activation and Bim induction in neuron death.</a> The Biochemical journal. 455 (1), 15-25 (2013).</li><li>Sotthibundhu, 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=Beta-amyloid(1-42)+induces+neuronal+death+through+the+p75+neurotrophin+receptor.">Beta-amyloid(1-42) induces neuronal death through the p75 neurotrophin receptor.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 28 (15), 3941-3946 (2008).</li><li>Kar, S., Quirion, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+peptides+and+central+cholinergic+neurons:+functional+interrelationship+and+relevance+to+Alzheimer's+disease+pathology.">Amyloid beta peptides and central cholinergic neurons: functional interrelationship and relevance to Alzheimer's disease pathology.</a> Progress in brain research. 145, 261-274 (2004).</li><li>Leranth, C., Frotscher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+septal+region+in+the+rat+brain:+cholinergic-GABAergic+interconnections+and+the+termination+of+hippocampo-septal+fibers.">Organization of the septal region in the rat brain: cholinergic-GABAergic interconnections and the termination of hippocampo-septal fibers.</a> The Journal of comparative neurology. 289 (2), 304-314 (1989).</li><li>Fa, 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=Preparation+of+oligomeric+beta-amyloid+1-42+and+induction+of+synaptic+plasticity+impairment+on+hippocampal+slices.">Preparation of oligomeric beta-amyloid 1-42 and induction of synaptic plasticity impairment on hippocampal slices.</a> Journal of visualized experiments : JoVE. (41), (2010).</li><li>Paxinos, G., Franklin, K. B. 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> The Mouse Brain in Stereotaxic Coordinates. , (2001).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of visualized experiments : JoVE. (65), (2012).</li><li>Currle, D. S., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+freezing+and+cryosectioning+E12.5+mouse+brain.">Flash freezing and cryosectioning E12.5 mouse brain.</a> Journal of visualized experiments : JoVE. (4), (2007).</li><li>Jin, 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=Soluble+amyloid+beta-protein+dimers+isolated+from+Alzheimer+cortex+directly+induce+Tau+hyperphosphorylation+and+neuritic+degeneration.">Soluble amyloid beta-protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (14), 5819-5824 (2011).</li><li>Lambert, M. 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=Diffusible,+nonfibrillar+ligands+derived+from+Abeta1-42+are+potent+central+nervous+system+neurotoxins.">Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (11), 6448-6453 (1998).</li><li>Masters, C. L., Selkoe, 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=Biochemistry+of+amyloid+beta-protein+and+amyloid+deposits+in+Alzheimer+disease.">Biochemistry of amyloid beta-protein and amyloid deposits in Alzheimer disease.</a> Cold Spring Harbor perspectives in medicine. 2 (6), a006262 (2012).</li><li>Chakrabarti, 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=Estrogen+receptor+agonists+for+attenuation+of+neuroinflammation+and+neurodegeneration.">Estrogen receptor agonists for attenuation of neuroinflammation and neurodegeneration.</a> Brain research bulletin. 109C, 22-31 (2014).</li><li>Cirrito, J. R., 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+assessment+of+brain+interstitial+fluid+with+microdialysis+reveals+plaque-associated+changes+in+amyloid-beta+metabolism+and+half-life.">In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 23 (26), 8844-8853 (2003).</li><li>Puzzo, 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=Picomolar+amyloid-beta+positively+modulates+synaptic+plasticity+and+memory+in+hippocampus.">Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 28 (53), 14537-14545 (2008).</li><li>Puzzo, 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=Endogenous+amyloid-beta+is+necessary+for+hippocampal+synaptic+plasticity+and+memory.">Endogenous amyloid-beta is necessary for hippocampal synaptic plasticity and memory.</a> Annals of. 69 (5), 819-830 (2011).</li><li>Akpan, 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=Intranasal+delivery+of+caspase-9+inhibitor+reduces+caspase-6-dependent+axon/neuron+loss+and+improves+neurological+function+after+stroke.">Intranasal delivery of caspase-9 inhibitor reduces caspase-6-dependent axon/neuron loss and improves neurological function after stroke.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 31 (24), 8894-8904 (2011).</li><li>Fulmer, C. G., 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=Astrocyte-derived+BDNF+supports+myelin+protein+synthesis+after+cuprizone-induced+demyelination.">Astrocyte-derived BDNF supports myelin protein synthesis after cuprizone-induced demyelination.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 34 (24), 8186-8196 (2014).</li><li>Thakker-Varia, S., 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=The+neuropeptide+VGF+is+reduced+in+human+bipolar+postmortem+brain+and+contributes+to+some+of+the+behavioral+and+molecular+effects+of+lithium.">The neuropeptide VGF is reduced in human bipolar postmortem brain and contributes to some of the behavioral and molecular effects of lithium.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 30 (28), 9368-9380 (2010).</li><li>Greferath, U., 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=Enlarged+cholinergic+forebrain+neurons+and+improved+spatial+learning+in+p75+knockout+mice.">Enlarged cholinergic forebrain neurons and improved spatial learning in p75 knockout mice.</a> The European journal of neuroscience. 12 (3), 885-893 (2000).</li><li>Brashear, H. R., Zaborszky, L., Heimer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+GABAergic+and+cholinergic+neurons+in+the+rat+diagonal+band.">Distribution of GABAergic and cholinergic neurons in the rat diagonal band.</a> Neuroscience. 17 (2), 439-451 (1986).</li><li>Clarke, 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=Cholinergic+innervation+of+the+rat+dentate+gyrus:+an+immunocytochemical+and+electron+microscopical+study.">Cholinergic innervation of the rat dentate gyrus: an immunocytochemical and electron microscopical study.</a> Brain research. 360 (1-2), 349-354 (1985).</li><li>Garcia-Osta, A., Alberini, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+mediates+memory+formation.">Amyloid beta mediates memory formation.</a> Learning &amp; memory. 16 (4), 267-272 (2009).</li></ol>

著者は、彼らが競合する金融利害関係がないことを宣言します。

成功したAβ1-42注入を達成するために、実験者または外科医は、必要があります：1）無菌技術を使用します。 2）正しく、正確な座標を持つ対象の脳領域を特定します。 3）適切にAPとML軸に水平に脳と定位フレームにマウスを確保することができます。 4）精度のマイクロマニピュレーターを操作する能力を有します。 5）適切な術後ケアを確保します。これらの重要なステップに従っ...

アミロイドβ（Aβ1-42）で構成されているアミロイド斑沈着は、アルツハイマー病（AD）の病理の重要な特徴です。多くの研究は、組換えオリゴマーAβ1-42の高いまたは毒性レベルが神経細胞死、シナプスジストロフィー、損失および機能障害を誘発することが示されています。ならびに学習および記憶の欠損1-4。影響を受けた脳領域は、海馬、皮質、及び前脳基底核と扁桃体の5,6のような皮質下構造を含みます。これまで、ADのAβ1-42病状をシミュレートしようとする多数のトランスジェニックマウスモデルが存在します。歪みに応じてこれらの動物は、ADの病理学的特徴の選択を検討するのに有用であることが判明しています。残念ながら、2トランスジェニックライン、APP23および5XFADを除いて、これらのマウスは完全にニューロン損失、ADの重要な側面を複製することはありません。でもAPP23と5XFAD、神経細胞死のobserで観察された神経細胞の損失でVEDは、微妙な年齢依存し、いくつかの選択領域7,8に単離しました。 野生型マウスの脳へのオリゴマーAβ1-42の直接注入はamyloidopathy 1,9,10の神経細胞死の側面を複製インビボモデルに優れたを提供します。一般的に利用するトランスジェニックマウスモデルとは異なり、オリゴマーAβ1-42注入モデルは、急性空間的、時間的な方法でAβ1-42のレベルを上昇させるのに最適です。このモデルのために、野生型マウスを使用する利点は、トランスジェニックマウスの系統に導入された変異の潜在的な補償または副作用がなくなります。過去の研究では、海馬に、Aβ1-42の毒性レベルを注入すると、1週間以内に1注射部位の近傍で神経細胞死を誘発することが示されています。また、Aβ1-42は、コリン作動性ニューロン11前脳基底部に毒性の観察と一致していること海馬に突出コリン作動性ニューロン（BFCN）集団は、効果的に脳内の孤立した神経回路の検査を可能にする、マウスにおけるβアミロイド注入1,10以下の7〜14日以内に20〜50％に減少します。大部分の制御/車両とオリゴマーAβ1-42溶液のための海馬12の歯状回に同側BFCNプロジェクトので比較は左右の半球1の間で行うことを可能にする脳のいずれかの側に注入することができます。 この報告では、成人の野生型C57BL / 6Jマウスの詳細な外科的および注射の方法論を提供します。このマウス株があるため、研究での広範な使用を選択されています。技術的には、いずれかの脳の領域は、しかし、ここで私たちは技術を説明するために、ターゲットとして、海馬の歯状回を使用し、注入のために標的とすることができます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ketamine HCl (100mg/ml)</td>
			<td>Henry Schein Medical</td>
			<td>1049007</td>
			<td>100 mg ketamine per 1 kg animal</td>
		</tr>
		<tr>
			<td>Xylazine (20mg/ml)</td>
			<td>Henry Schein Medical</td>
			<td>not available</td>
			<td>10 mg xylazine per 1 kg animal</td>
		</tr>
		<tr>
			<td>Buprenex (0.3mg/ml)</td>
			<td>Henry Schein Medical</td>
			<td>1217793</td>
			<td>0.1 mg buprenex per 1 kg animal</td>
		</tr>
		<tr>
			<td>A&#946;1-42</td>
			<td>David Teplow/UCLA</td>
			<td>not available</td>
			<td>100 &#956;M; This amyloid was used in the paper</td>
		</tr>
		<tr>
			<td>A&#946;1-42</td>
			<td>Bachem</td>
			<td>H-1368</td>
			<td>Can be used in place of amyloid from the Teplow lab</td>
		</tr>
		<tr>
			<td>A&#946;1-42</td>
			<td>American Peptide</td>
			<td>62-0-80B</td>
			<td>Can be used in place of amyloid from the Teplow lab</td>
		</tr>
		<tr>
			<td>Scrambled A&#946;1-42</td>
			<td>American Peptide</td>
			<td>62-0-46B</td>
			<td>Can be used as control peptide for comparing A&#946;1-42</td>
		</tr>
		<tr>
			<td>NU4 Antibody (Oligomeric Amyloid Antibody)</td>
			<td>Gift from William Klein/Northwestern U.</td>
			<td>not available</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>Anti-Amyloid Oligomeric Antibody&#160; (Polyclonal Rabbit)</td>
			<td>EMD Millipore</td>
			<td>AB9234</td>
			<td>May be used in place of Nu4; needs to&#160; be tested by the end user</td>
		</tr>
		<tr>
			<td>6E10 Antibody (Monoclonal Mouse) (Amyloid Antibody)</td>
			<td>Biolegend</td>
			<td>sig-39320</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>ChAT Antibody (Polyclonal Goat)</td>
			<td>Millipore</td>
			<td>AB144P</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>DeadEnd Fluorometric TUNEL system</td>
			<td>Promega</td>
			<td>G3250</td>
			<td>Follow manufacturer&#39;s directions for use</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>Use when coverslipping slides</td>
		</tr>
		<tr>
			<td>Fluorogold</td>
			<td>Fluorochrome, LLC</td>
			<td>not available</td>
			<td>2% solution</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (200 proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-4</td>
			<td>For making 70% ethanol for sanitizing and disinfecting</td>
		</tr>
		<tr>
			<td>Novex 10-20% Tricine gel</td>
			<td>Life Technologies</td>
			<td>EC6625BOX</td>
			<td>For separating A&#946;1-42</td>
		</tr>
		<tr>
			<td>Novex Tricine SDS Running Buffer (10X)</td>
			<td>Life Technologies</td>
			<td>LC1675</td>
			<td>For running 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>Novex Tris-Glycine Transfer Buffer (25X)</td>
			<td>Life Technologies</td>
			<td>LC3675</td>
			<td>For transferring 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>SuperSignal Western Blot Enhancer</td>
			<td>Thermo Scientific</td>
			<td>46640</td>
			<td>For enhancing A&#946;1-42 signal; follow manufacturer&#39;s protocol</td>
		</tr>
		<tr>
			<td>Protran BA79 Nitrocellulose Blotting Membrane, 0.1 &#956;m</td>
			<td>GE Healthcare Life Sciences</td>
			<td>10402088</td>
			<td>For transferring 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>Xcell SureLock Mini-Cell</td>
			<td>Life Technologies</td>
			<td>EI0001</td>
			<td>Electrophoresis aparatus for running 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>GenTeal Lubricant Eye Gel</td>
			<td>Novartis</td>
			<td>not available</td>
			<td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores</td>
		</tr>
		<tr>
			<td>Refresh Optive Lubricant Eye Drops</td>
			<td>Allergan</td>
			<td>not available</td>
			<td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores; Can be used in place of GenTeal</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Stoelting</td>
			<td>50998</td>
			<td>For sanitizing and disinfecting</td>
		</tr>
		<tr>
			<td>Round/Tapered Spatula&#160;</td>
			<td>VWR</td>
			<td>82027-490</td>
			<td>For opening animal mouth</td>
		</tr>
		<tr>
			<td>Bulldog Serrefines Clamps (Jaw Dims. 9X1.6mm; Length 28mm)</td>
			<td>Fine Science Tools</td>
			<td>18050-28</td>
			<td>Optional; For keeping scalp skin apart during injection</td>
		</tr>
		<tr>
			<td>Straight Fine Scissors (Cutting edge 25mm; Length 11.5cm)</td>
			<td>Fine Science Tools</td>
			<td>14060-11</td>
			<td>For cutting scalp</td>
		</tr>
		<tr>
			<td>#3 Scalpel Handle</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>#11 Surgical Blade</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td>For making scalp incision</td>
		</tr>
		<tr>
			<td>Student Standard Pattern Forcep (Tip Dims. 2.5x1.5mm; Length 11.5cm)</td>
			<td>Fine Science Tools</td>
			<td>91100-12</td>
			<td>For holding scalp closed during suturing</td>
		</tr>
		<tr>
			<td>Trimmer Combo Kit</td>
			<td>Kent Scientific</td>
			<td>CL9990-1201</td>
			<td>For shaving hair</td>
		</tr>
		<tr>
			<td>T/Pump Warm Water Recirculator&#160;</td>
			<td>Kent Scientific&#160;</td>
			<td>TP-700</td>
			<td>For warming animal during surgery</td>
		</tr>
		<tr>
			<td>Resusable Warmining Pad (5&#34; x 10&#34;)</td>
			<td>Kent Scientific&#160;</td>
			<td>TPZ-0510FEA</td>
			<td>For attaching it to the T/Pump warm water recirculator to warm the animal during surgery</td>
		</tr>
		<tr>
			<td>Cordless Micro Drill</td>
			<td>Stoelting</td>
			<td>58610</td>
			<td>Use 0.8mm steel burrs to drill holes in the skull</td>
		</tr>
		<tr>
			<td>Lab Standard Stereotaxic Instrument with Mouse &#38; Neonatal Rat Adaptor</td>
			<td>Stoelting</td>
			<td>51615</td>
			<td></td>
		</tr>
		<tr>
			<td>Just for Mouse Stereotaxic Instrument</td>
			<td>Stoelting</td>
			<td>51730</td>
			<td>Can use this in place of Stoelting Cat. #51615</td>
		</tr>
		<tr>
			<td>Quintessential Stereotaxic Injector</td>
			<td>Stoelting</td>
			<td>53311</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Glass Bead Sterilizer</td>
			<td>Stoelting</td>
			<td>50287</td>
			<td>For sterilizing stainless steel instruments</td>
		</tr>
		<tr>
			<td>Sterile Surgical Drape (18&#34; x 26&#34;)</td>
			<td>Stoelting</td>
			<td>50981</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton Syringe 50 ml, Model 705 RN SYR, NDL</td>
			<td>Hamilton Company</td>
			<td>7637-01</td>
			<td>For brain injection; use different syringes for different solutions</td>
		</tr>
		<tr>
			<td>29 Gauge Needle, Small Hub RN NDL</td>
			<td>Hamilton Company</td>
			<td>7803-06</td>
			<td>For attaching to the Hamilton syringe for brain injection</td>
		</tr>
		<tr>
			<td>1 ml BD Tuberculin Syringes</td>
			<td>VWR</td>
			<td>BD309659</td>
			<td>For administering anesthesia and saline</td>
		</tr>
		<tr>
			<td>30 Gauge Needle (0.5&#34;)</td>
			<td>VWR</td>
			<td>BD305106</td>
			<td>For administering anesthesia and saline</td>
		</tr>
		<tr>
			<td>Portable Electronic CS Series Scale (Ohaus)</td>
			<td>VWR</td>
			<td>65500-202</td>
			<td>For weighing animals to determine anesthesia dose</td>
		</tr>
		<tr>
			<td>Hot plate (Top Plate Dims. 7.25x7.25in)</td>
			<td>VWR</td>
			<td>47751-148</td>
			<td>For warming animals post-surgery</td>
		</tr>
		<tr>
			<td>Sofsilk Silk Suture C-1 Cutting 3/8, 12 mm</td>
			<td>Covidien</td>
			<td>S1173</td>
			<td>For closing wound</td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive (3M)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-361931</td>
			<td>Optional: for aiding in wound closure; Use with suture.</td>
		</tr>
		<tr>
			<td>Cotton-Tipped Wooden-Shaft Sterile Applicators</td>
			<td>Fisher scientific</td>
			<td>22-029-488</td>
			<td>For cleaning and drying surgical wound</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-550-15</td>
			<td>For collecting brain sections</td>
		</tr>
		<tr>
			<td>VWR Micro Cover Glass 24 X 50 mm</td>
			<td>VWR</td>
			<td>48393241</td>
			<td>For mounting microscope slides</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nalgene Syringe Filter 0.2 &#956;m</td>
			<td>Fisher Scientific</td>
			<td>194-2520</td>
			<td>For sterilizing saline solution</td>
		</tr>
		<tr>
			<td>Sterile dual tip skin markers by Viscot Medical</td>
			<td>Medline</td>
			<td>VIS1422SRL91</td>
			<td>For marking coordinates on the skull</td>
		</tr>
	</tbody>
</table>

stereotaxic infusion of oligomeric amyloid-beta into the mouse hippocampus

アルツハイマー病は、高齢化に影響を与える神経変性疾患です。疾患の重要な神経病理学的特徴は、アミロイドβの過剰産生と罹患個体の脳領域におけるアミロイドβ斑の沈着です。年間を通して、科学者は、アミロイドベータ病理を複製しようとする多数のアルツハイマー病のマウスモデルを生成しました。残念ながら、マウスモデルは、選択的に、疾患の特徴を模倣します。神経細胞死、アルツハイマー病患者の脳における顕著な効果は、顕著に、これらのマウスに欠けています。ニューロンに最も毒性であることが証明されているアミロイドβの形 - - 定位脳へしたがって、私たちと他の人が直接アミロイドβの可溶性オリゴマー種を注入する方法を採用しています。このレポートでは、選択脳領域におけるアミロイドベータレベルを増加させるこの手術法を文書化するために雄のC57BL / 6Jマウスを利用しています。ザ注入ターゲットは、コリン作動性回路によって接続されている前脳基底部と一緒に、この脳の構造ため、海馬の歯状回である疾患における変性の領域のいずれかを表します。細胞死における同時増加とブロカの対角バンドの垂直肢におけるコリン作動性ニューロンの損失がある間定位注入を介して歯状回におけるアミロイドβの上昇の結果は、1週間以内に歯状回におけるニューロン損失の増加を明らかに前脳基底部の。これらの効果は、2週間まで観察されます。我々のデータは、現在のアミロイドβ注入モデルは、短期的に特定の領域のニューロン死に対処するための代替的なマウスモデルを提供することを示唆しています。このモデルの利点は、アミロイドベータが、空間的および時間的な方法で上昇させることができるということです。

ヒト組換えオリゴマーAβ1-42の調製本発明の方法は、モノマー、ダイマー、トリマー、およびテトラマー（ 図1A）からなる可溶性オリゴマー種をもたらします。これらの低分子量のAβ1-42の種ではなく、原線維およびプラークは、ニューロン1,4,9,17-19に最も毒性であることが、多数の設定で示されています。オリゴマーAβ1-42は、マウスの脳内の神経?...

Watch this Scientific Journal Video about Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus at JoVE.com

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

マウス海馬にオリゴマーアミロイドベータの定位注入

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

stereotaxic-infusion-oligomeric-amyloid-beta-into-mouse

Research

JoVE Journal

Neuroscience

18.0K ビュー.  Columbia University Medical Center. Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

ビデオ: Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

超音波ガイド下のマウスの前脳へのマイクロインジェクション子宮内で E9.5で

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

神経科学

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

大人のラットの特定の脳領域の興奮毒性病変に対する定位手術

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作<em&gt;子宮内で</em&gt;エレクトロポレーション

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

マウス脊髄における条件遺伝子操作のためのウイルスベクターの定位注射

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Building An Open-source Robotic Stereotaxic Instrument

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for around $1,000 (excluding a drill), using industry standard stepper motors and CNC controlling software. Each axis has variable speed control and may be operated simultaneously or independently. The robot&#39;s flexibility and open coding system (g-code) make it capable of performing custom tasks that are not supported by commercial systems. Its applications include, but are not limited to, drilling holes, sharp edge craniotomies, skull thinning, and lowering electrodes or cannula. In order to expedite the writing of g-coding for simple surgeries, we have developed custom scripts that allow individuals to design a surgery with no knowledge of programming. However, for users to get the most out of the motorized stereotax, it would be beneficial to be knowledgeable in mathematical programming and G-Coding (simple programming for CNC machining).
The recommended drill speed is greater than 40,000 rpm. The stepper motor resolution is 1.8&#176;/Step, geared to 0.346&#176;/Step. A standard stereotax has a resolution of 2.88 &#956;m/step. The maximum recommended cutting speed is 500 &#956;m/sec. The maximum recommended jogging speed is 3,500 &#956;m/sec. The maximum recommended drill bit size is HP 2.

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (computer numeric controlled; CNC) stereotaxic instrument for around $1,000 (excluding a drill).

オープンソースロボット定位固定装置を構築する

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

CNSへの標的化物質送達のためのマウス顕微注入法<i&gt;経由で</i&gt;内頸動脈

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

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, ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

行動中マウスの海馬における振動を制限空間録音

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

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 ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).