This study was supported by the National Health and Medical Research Council of Australia (NHMRC) Project grant 1022839. AJH is an Australian Research Council (ARC) FT3 Future Fellow (FT100100835). The Florey Institute of Neuroscience and Mental Health acknowledges support from the Victorian Government&#8217;s Operational Infrastructure Support Grant.

注：動物のすべての実験手順は、神経科学·精神衛生動物倫理委員会のフローリー研究所によって承認され、オーストラリアの国立保健医療研究評議会に準拠しては、科学的な目的のための動物の管理と使用の実践のためのコード（ 第7版を発行2004）。 1.環境マニピュレーション<ol><li>男女ペアリング<ol><li>性的に成熟した（&gt; 8週齢）、年齢をマッチさせた雄と雌のマウスを使用してください。 注：私たちは、一般的に、C57BL / 6マウスを使用しますが、このプロトコルでスイスマウスを使用して、同じ成果があった。我々は、典型的にはn =（n = 4の男性）は、n = 2オス - オスのペアに分け、各実験についてはn = 8の男性であり、n = 8の雌を使用して2メス - メスのペアた（n = 4匹）、そしてn = 4男女ペア（n = 4の男性であり、n = 4匹）。 </li><li>動物保持施設、グループ社内に到着すると&gt; 3日間、男女別のマウスは、順応する。こことthrougハウトは男女ペアリング、余分な環境刺激を維持する（ 例えば 、周囲温度、光：暗サイクル、食料、水、取り扱いおよびクリーニング）定数またはすべてのマウスに等しく分配する。 </li><li>ランダムにオス - オスペア、メス - メスのペア、または男女ペアに各マウスを割り当てます。食物および水を自由摂取アクセスを分離してきれいなケージ（2匹/ケージ）に各ペアを置き、単純に7日間連続して、この方法でそれらを収容する。 注：ルーチン畜産は、この期間中にOKですが、すべてのマウスに均等に分配されなければならない。男性と女性の同腹仔のペアリングを避けるように注意してください。 </li></ol></li><li>環境強化<ol><li>性的に成熟した（&gt; 8週齢）、年齢をマッチさせた雄または雌のマウスを使用してください。 注：私たちは、一般的にはn = 6 /治療群に分け、各実験のためのn = 18マウスを使用します。 </li><li>動物保持施設に到着すると、グループに収容され、同一規格内に収容された（SH）（ 例えばそれらを保つ</em&gt; 3日間は順応さのために&gt;この段階で）以下の条件を参照してください。ここでは、実験を通して、余分な環境刺激を維持する（ 例えば 、周囲温度、光：暗サイクル、食料、水、取り扱いおよびクリーニング）定数またはすべてのマウスに等しく分配する。 </li><li>環境エンリッチメントを開始するには、無作為に3グループのいずれかに各マウスを割り当てます。SH。実行中のホイール（RW）、または環境濃縮（EE）と一緒に食物および水に自由に摂取と同じ清潔なケージにマウスの各群を配置。 注：すべてのグループのためにここでは、より大きなラット保持ケージ42センチ、幅の広い27センチメートルを測定し、深い16センチメートルを使用した。 <ol><li>また、各グループに以下の環境条件を提供する：床にのみゴミを含んSH（紙ペレットまたはおがくず） SHプラス2走行輪を備えRW。これで元にRWプラス玩具（ロープ、はしご、トンネル、及びそのような空のペーパータオルロールとティッシュペーパーの破片などのオブジェクト）を含むEEplore、遊び、登る、非表示、および巣。 </li><li> 14日間連続して、この方法でそれらを家。 注：ルーチン畜産は、この期間中にOKですが、すべてのマウスに均等に分配されなければならない。 </li></ol></li><li> 1時間/日（同時間毎日）、5日/週のための新規のおもちゃを含むより大きなケージにそれらを一緒に配置することで、追加の「超濃縮&#39;（SE）に従うことを条件EEマウス。 注：ここでは46センチ、幅の69センチメートル長く、そして40センチメートル深いプラスチック浴槽を使用した。 <ol><li>おもちゃの異なるセット各セッションを提示することによって新規性を維持します。香りを除去するために石鹸水と80％エタノールを有するマウスに再提示されるクリーンな玩具。 </li><li>各SEセッションに続いて、彼らのEEケージにマウスを返す。ハンドルSHとRWマウスこの期間を通じて（SE自体を除く）SEマウスと同じ（ 例えば削除してから、そのケージに各SHおよびRWマウスを返す）。 </li></ol></li></ol></li></ol><ol><li>準備<ol><li>市販されている無菌の浸透圧ポンプと脳注入キットを、使用してください。移植前日、無菌技術を使用して、各ポンプの充填（ポンプの取扱説明書に記載されている）の無菌薬剤または車両溶液とチューブとカニューレを接続することにより、プライムインプラント。それらを一緒に接続し、37℃で一晩滅菌生理食塩水でインキュベートする。交差汚染を避けるために、別々に薬物およびビヒクルで満たされた移植片をインキュベートする。 </li></ol></li><li>手術。 注：実験者の国によっては、特殊な動物実験の許可または引当金は、手術や術後の実験のようなタイプを追求するために必要とされている。 <ol><li>すべての手術用機器を殺菌し、全体で無菌技術を採用しています。 （ 例えば 、空気中で1〜2％イソフルランを使用して）、マウスを麻酔し、ステールに入れてくださいotaxicヘッドフレーム。作業中は麻酔の深さを確認し、必要に応じて、さらに麻酔薬を投与し（有害な足のピンチに手足離脱の例がないことは、十分な麻酔の指標である）。確実に眼を潤滑眼軟膏による乾燥から保護し、そしてマウスの体温は、手順全体を通して通常のままであることをしている。 </li><li>目の間から開始し、頭蓋骨の後縁に2cmの後部を終え、皮膚を通して正中切開を行います。ブラントは、（接続管が完了時に曲げるべきではありません）皮下「ポケット」カニューレが移植された後に、快適にポンプ及び連結チューブにフィットするのに十分な大きさを作成するために離れてマウスの背部の下、基礎となる筋膜から皮膚を解剖。頭蓋骨の背側面上から筋膜をクリアこすり。 </li><li>約1.5ミリメートルの直径歯科用バーを使用して、適切な定位で頭蓋骨にドリルダウンすると、座標until骨の薄い、柔軟な層が残る。ピール離れて細かい鉗子を使用して、この層（これは歯科用バーで、基礎となる硬膜と脳の損傷を避ける）。頭蓋骨は、任意の血液と骨の断片の清掃、それ以上の出血がないことをされていることを確認。 </li><li>カニューレホルダに保持されたカニューレを使用すると、マウスの背中の上に重なる皮下「ポケット」にポンプと接続管を配置。次に、適切な定位座標で、脳の表面にカニューレの先端を配置します。慎重に脳へのカニューレを下げるが、必要な深さの短い約1ミリメートルを停止します。 注：残りの深さは、歯科アクリルの第一層の適用後にネゴシエートされます。 </li><li>頭蓋骨の表面が清潔で乾燥している再確認し、その後、歯科アクリルの小さなボリュームを準備し、頭蓋骨の全露出領域の上にそれを滑らかにするためにつまようじを使用し、目の周りの頭蓋骨を通してバリの穴に含むEカニューレ。アクリルが硬化する前に、カニューレがターゲットに残りの深さを傾ける下げる。 </li><li>歯科アクリルの別の小さなボリュームを準備し、所定の位置にカニューレを固定するために、第1の過だけでなく、カニューレのための白いプラスチックの支持体上にこのレイヤ。必要に応じてアクリルの追加の層を繰り返します。 </li><li>アクリルが硬化した後は、慎重にカニューレホルダーを取り外し、ブタン火炎で加熱さメスの刃を使用して白いプラスチック取り外し可能なカニューレタブを遮断する。最後に、インプラント全体にわたって閉じた皮膚を縫合。 </li></ol></li><li>動物の手術後の治療<ol><li>皮膚の余白に消毒軟膏を適用し、マウスに抗炎症を管理する（ 例えばメロキシカム、3 mg / kgを皮下）。 、フレームからマウスを外し加熱ランプ下に置き、そしてそれが意識を取り戻すまで観察する。完全に回復するまで、他の動物の会社に手術を受けたマウスを返さないでください。 注：EXPそのような男女のペアリングと環境充実などerimental操作が開始されるか、または早くとも手術翌日再開することができます。 </li><li>自分の体重、毎日の一般的な動作と外観を監視します。局所消毒軟膏と外科的切開の周りに感染の兆候（ 例えば腫脹、発赤、膿）を扱います。必要に応じて、全身鎮痛剤および/ ​​または抗生物質で（&gt; 10％の体重、引きこもり、グルーミングの欠如、「毛羽立ち」、運動機能障害、発作の例えば損失）兆候の病気、痛み、ストレスや不快感を扱います。 </li><li> &gt; 15％の体重減少や​​病気、痛み、ストレスや不快感の症状は、是正処置の1週間以内に回復不能な場合、マウスを安楽死させる。 </li></ol></li></ol> 3.脳組織の調製、免疫組織化学的処理、および立体学<ol><li>かん流<ol><li>すぐに環境/薬物の操作以下、研究のために脳を準備します。 <br/&gt;注：ここに報告する実験ではSNcのTH +ニューロンの数を、異なる処置群のそれぞれにおいて測定して比較した。 </li><li>まずマウス（ 例えば 、100 mg / kgを腹腔内ペントバルビタールナトリウム）を殺すために麻酔薬の過剰摂取を管理する。一度麻酔し、心臓が鼓動を停止する前に、その背中の上でマウスを置くとネクタイまたは両方前肢を突き止める。 </li><li>胸部を覆う皮膚を切り取るメスを使用すると、その後腹腔内にちょうど肋骨の下の筋肉を通して切開する。胸郭の剣状突起上のクランプを配置し、振動板を露出する肝臓から持ち上げて胸郭を持ち上げます。 </li><li>胸郭、肺および心臓を露出させるために頭の上に折り返さできるまで、絞りを介して、大きなハサミを使用して、両側の横方向リブを切断する。細かいハサミを使用すると、左のventriのベースを切断、血液と灌流ソリューションの出口点として右心房に小さな切開を作るクレと左心室、左心房を通ってカニューレを配置し、大動脈に。 </li><li>ヘパリン化温かい（37℃）ポンプその後適所にカニューレを固定し、右心房を出る溶液まで、血管系を介して0.1 Mリン酸緩衝生理食塩水（PBS）は、血液が完全に自由である。全体マウスがうまく固定されるまで、次に、血管系を通って（例えば、PBS中の4％パラホルムアルデヒドとして）冷たい（4℃）固定液ポンプ。脳のシンクまで、2〜3日間30％ショ糖を含むPBS中で脳と場所を削除します。 注：組織調製の他のタイプは、対応する研究室の専門知識に応じて適用される場合があります。 </li></ol></li><li>フリーフローティング凍結切片の調製<ol><li>関心の脳領域を通じて連続切片をカットし、PBSでこれらを集める。 注：私たちは、クライオスタットを用いて、40μmの厚さの切片をカット。組織切片の他のタイプはcorrespondiの専門知識に応じて適用されることがありますNGの研究室。 </li></ol></li><li>免疫組織化学<ol><li>目的のタンパク質のための標準的な免疫組織化学を実行します。 48時間4℃で：（400 1）は、ポリクローナル、ビオチン化ヤギ抗ポリクローナルウサギ抗TH、次いで、30分間免疫反応を室温でPBS中の5％正常ヤギ血清および0.3％トリトンX-100で切片をインキュベート2時間室温で：-rabbit（千1）。 </li><li> 18〜20分間、次いで室温でコバルト及びニッケル強化ジアミノベンジジン（0.5mg / ml）をで、1時間、室温で;次に、アビジン - ペルオキシダーゼ（1：500）でインキュベートジアミノベンジジンインキュベーションの最後の3~5分間クロマゲン析出を触媒するために過酸化水素（0.01％）を加える。後に、上記の各ステップの間、セクションの前にPBSで10分間3回洗浄する。 </li><li>ゼラチン化顕微鏡スライド上のセクションをマウントし、空気それらを乾燥し、ニッスル染色（ニュートラルレッド）、アルコール脱水、（X-3B）をクリアし、カバースリップ。 </li></ol></li><lI&gt;立体学<ol><li>公平な立体的な方法を用いて、TH +とTH-のSNc（とVTAおよびLC）ニューロンの合計数を推定。 stereologistが受けた治療に盲目であることを確認してください。相馬直径&lt;5ミクロンに基づいてグリアを除外し、目に見える核を有する細胞のみをカウントします。 </li><li> TH +細胞およびPaxinosとワトソン7の脳地図によると、解剖学的ランドマーク/境界の空間的な位置によってのSNc（とVTAおよびLC）を特定します。 （; X = 100ミクロン、Y = VTAとLCのための100μmのX = Y140μmで、= SNCの140μm）を定期的に予め定められた間隔でカウントフレーム内のTH +細胞（55 X 55ミクロン= 3025ミクロン2）をカウントすべての4番目のセクション内の各核全体に。 </li></ol></li></ol>

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L., Joh, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transneuronal+regulation+of+tyrosine+hydroxylase+expression+in+olfactory+bulb+of+mouse+and+rat.">Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 3, 69-78 (1983).</li><li>Black, I. B., Chikaraishi, D. M., Lewis, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-synaptic+increase+in+RNA+coding+for+tyrosine+hydroxylase+in+a+rat+sympathetic+ganglion.">Trans-synaptic increase in RNA coding for tyrosine hydroxylase in a rat sympathetic ganglion.</a> Brain research. 339, 151-153 (1985).</li><li>Zigmond, R. E., Chalazonitis, A., Joh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preganglionic+nerve+stimulation+increases+the+amount+of+tyrosine+hydroxylase+in+the+rat+superior+cervical+ganglion.">Preganglionic nerve stimulation increases the amount of tyrosine hydroxylase in the rat superior cervical ganglion.</a> Neuroscience letters. 20, 61-65 (1980).</li><li>Biguet, N. F., Rittenhouse, A. R., Mallet, J., Zigmond, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preganglionic+nerve+stimulation+increases+mRNA+levels+for+tyrosine+hydroxylase+in+the+rat+superior+cervical+ganglion.">Preganglionic nerve stimulation increases mRNA levels for tyrosine hydroxylase in the rat superior cervical ganglion.</a> Neuroscience letters. 104, 189-194 (1989).</li><li>Richard, F., 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=Modulation+of+tyrosine+hydroxylase+gene+expression+in+rat+brain+and+adrenals+by+exposure+to+cold.">Modulation of tyrosine hydroxylase gene expression in rat brain and adrenals by exposure to cold.</a> Journal of neuroscience research. 20, 32-37 (1988).</li><li>Schalling, M., Stieg, P. E., Lindquist, C., Goldstein, M., Hokfelt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+increase+in+enzyme+and+peptide+mRNA+in+sympathetic+ganglia+after+electrical+stimulation+in+humans.">Rapid increase in enzyme and peptide mRNA in sympathetic ganglia after electrical stimulation in humans.</a> Proc Natl Acad Sci U S A. 86, 4302-4305 (1989).</li><li>Liaw, J. J., He, J. R., Barraclough, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+changes+in+tyrosine+hydroxylase+mRNA+levels+in+A1,+A2+and+locus+ceruleus+neurons+following+electrical+stimulation+of+A1+noradrenergic+neurons.">Temporal changes in tyrosine hydroxylase mRNA levels in A1, A2 and locus ceruleus neurons following electrical stimulation of A1 noradrenergic neurons.</a> Brain research. Molecular brain research. 13, 171-174 (1992).</li><li>Watabe-Uchida, M., Zhu, L., Ogawa, S. K., Vamanrao, A., Uchida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-brain+mapping+of+direct+inputs+to+midbrain+dopamine+neurons.">Whole-brain mapping of direct inputs to midbrain dopamine neurons.</a> Neuron. 74, 858-873 (2012).</li><li>Tepper, J. M., Lee, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+control+of+substantia+nigra+dopaminergic+neurons.">GABAergic control of substantia nigra dopaminergic neurons.</a> Progress in brain research. 160, 189-208 (2007).</li><li>Hannan, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+and+brain+repair:+harnessing+the+therapeutic+effects+of+cognitive+stimulation+and+physical+activity+to+enhance+experience-dependent+plasticity.">Environmental enrichment and brain repair: harnessing the therapeutic effects of cognitive stimulation and physical activity to enhance experience-dependent plasticity.</a> Neuropathology and applied neurobiology. 40, 13-25 (2014).</li><li>Nithianantharajah, J., Hannan, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enriched+environments,+experience-dependent+plasticity+and+disorders+of+the+nervous+system.">Enriched environments, experience-dependent plasticity and disorders of the nervous system.</a> Nature reviews. Neuroscience. 7, 697-709 (2006).</li><li>Chmiel, D. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-environment+interactions+and+construct+validity+in+preclinical+models+of+psychiatric+disorders.">Gene-environment interactions and construct validity in preclinical models of psychiatric disorders.</a> Progress in neuro-psychopharmacology &amp; biological psychiatry. 35, 1376-1382 (2011).</li><li>Van de Weerd, H. A., Van Loo, P. L. P., Van Zutphen, L. F. M., Koolhaas, J. 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The authors have nothing to disclose.

環境の操作 これらの環境の操作（男女ペアリングと環境エンリッチメント）の設計の背後にある動機は、環境かどうかを決定することであった、かつ/または環境によってプロンプト動作は、中脳DAニューロンの数の変化と関連している。焦点は、環境を提供し、中脳DAシグナリングに係合する可能性がある行動を刺激する上でしたので。これらは、反対の性別と?...

中脳の腹側被蓋領域（VTA）と黒質緻密部（SNC）におけるニューロンによるDArgicシグナリングは報酬やる気認知、感情、モーター行動のために重要であると考えられている。しかし、多すぎたり少なすぎたり、中脳DAシグナリングは、神経学的障害（ 例えば 、パーキンソン病、注意欠陥多動性障害、統合失調症、および薬物嗜癖）の様々な多くの無効症状を引き起こす。増加またはこれらの症状を緩和シグナルDAを減少させる薬物には、しかし、彼らはまた、調節不全シグナリングおよびオフターゲット効果に起因する副作用を生じる。薬物の有効性はまた、脳の応答を代償に起因する経時的に低下する。課題は、したがって、よりター​​ゲットと生理方法で正常な脳DAシグナリングを復元することで、有利なアプローチは、増加または中脳DAニューロンの数を減少させることによってである。 証拠は、SEVのために蓄積してきた成熟した大人の細胞ではDAと他のカテコールアミン代謝および輸送に関与する遺伝子やタンパク質の発現が（1にレビュー）変更可能であることをERAL十年。 THの免疫陰性（TH-）ニューロンの数は逆のパターン（ すなわち増加を示している中脳では、チロシンヒドロキシラーゼ免疫陽性（TH +、DA合成における律速酵素）ニューロンの数は、次の神経毒政権2,3を増加減少その後3）を減少させる。これは、いくつかの細胞による「DA表現型」の利得その後損失と一致している。 TH +およびTH-のSNcニューロンの数は、これらのセル4,5の電気的活性を変化させる種々の処理、以下、等しいが反対方向に変化することが示されている。例えば、小コンダクタンスの注入、カルシウム活性化カリウム2週間の脳に（SK）チャネルアンタゴニストアパミン（同じ量）TH +と増加数が減少νTH-のSNcのmberは4,5ニューロン。対照的に、SKチャネルアゴニスト1-EBIOの注入は、TH +の数を増加させ、（同じ量）TH-のSNcニューロン4,5の数を減少させる。同様の変化が求心性入力4を対象としたいくつかの含むのSNcニューロンの活動を、ターゲットに様々なトリートメント後に見られた。神経活動と求心性入力によるSNcのDArgicニューロンの数のこの明らかな規制は、環境や行動SNCのニューロンの数に影響を与えることができる可能性を高める。異なる環境にさらされる実際成体マウスは、多かれ少なかれ中脳（のSNcおよびVTA）TH +ニューロン、およびこれらの環境による変化の少なくともいくつかの脳6におけるシナプス入力の同時遮断によって廃止されています。この通信の目的は以下のとおりである。（1）当社の環境操作および薬物注入を実装する方法についてさらに詳しく説明します。 （2）電子という我々の主張を支持するさらなるデータを提供するnvironmentは求心性入力を介して 、中脳DAニューロンの数を調節する。

<table border="0" cellpadding="0" cellspacing="0" width="1300">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Name of Material/ Equipment</td>
			<td style="width:636px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Isofluorane</td>
			<td style="width:636px;">Baxter Healthcare Pty Ltd, Baxter Drive, NSW 2146, Australia</td>
			<td style="width:119px;">AHN3640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">ALZET Osmotic pump 1002</td>
			<td style="width:636px;">DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td>
			<td>0004317</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">ALZET Brain infusion kit 1</td>
			<td style="width:636px;">DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td>
			<td>0004760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">ALZET cannula holder 1</td>
			<td style="width:636px;">DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td>
			<td>0008860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Vertex Monomer Self-curing (dental acrylic solvent)</td>
			<td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Vertex Self Curing (dental acrylic powder)</td>
			<td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">METACAM (Meloxicam)</td>
			<td>Troy Laboratories, 98 long Street, smithfield NSW 2164 Australia</td>
			<td style="width:119px;">L10100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Sodium Pentobarbitone</td>
			<td>Lethabarb, Virbac, Milperra, NSW, Australia</td>
			<td style="width:119px;">571177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Normal goat serum</td>
			<td>chemicon-temecula, CA</td>
			<td style="width:119px;">S26-Litre</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Triton X-100</td>
			<td>Merck Millipore Headquarters&#160;, 290 Concord road, Billerica, MA 01821</td>
			<td style="width:119px;">1.08603.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Polyclonal rabbit anti-tyrosine hydroxylase</td>
			<td>Merck Millipore Headquarters&#160;, 290 Concord road, Billerica, MA 01821</td>
			<td style="width:119px;">AB152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyclonal biotinylated goat anti-rabbit</td>
			<td>Dako Australia Pty. Ltd., Suite 4, Level 4, 56 Berry street, North Sydney, NSW, Australia 2060</td>
			<td style="width:119px;">EO432</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Avidin peroxidase</td>
			<td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td>
			<td style="width:119px;">A3151-1mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diamino-benzidine</td>
			<td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td>
			<td style="width:119px;">D-5637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereo Investigator</td>
			<td>MicroBrightField Bioscience, 185 Allen Brook Lane, Suite 101, Williston, VT 05495</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

environmental modulations of the number of midbrain dopamine neurons in adult mice

脳や「脳の可塑性」で長持ちの変化は、疾患または損傷後の適応行動と脳の修復を根底にある。さらに、我々の環境との相互作用は、脳の可塑性を誘導することができる。ますます、研究は、脳および行動障害を治療するための有益な脳の可塑性を刺激する環境を識別しようとしている。二つの環境の操作は、増加または成体マウスの脳におけるチロシンヒドロキシラーゼ免疫陽性（TH +、ドーパミンの律速酵素（DA）の合成）ニューロンの数を減少させるが記載されている。第一は、ペアリングの男性と一緒に継続的に男性で約12％、中脳のTH +ニューロンが増加し1週間、のための雌マウスを含むが、女性では約12％、中脳のTH +ニューロンを減少させる。第二は、私は、走行輪、おもちゃ、ロープ、ネスティング材料などを含む「濃縮環境 &#39;（EE）で2週間連続して住宅マウスを含み、男性では約14％、中脳のTH +ニューロンをncreases。さらに、プロトコルは、同時に環境的に誘発される脳の可塑性のメカニズムを識別しやすくするためにこれらの環境の操作中に、中脳に直接薬剤を注入するために記載されている。例えば、より中脳のTH +ニューロンのEE-誘導は中脳の神経細胞へのシナプス入力の同時遮断によって廃止されている。一緒に、これらのデータは、環境に関する情報は、「DA」の遺伝子の発現をオンまたはオフに切り替えるために、中脳のニューロンにシナプス入力を介して中継されることを示している。このように、基礎となるメカニズムを標的と適切な環境刺激、または薬物は、脳DAの不均衡に関連する脳や行動障害を治療するのに役立つかもしれない（ 例えば 、パーキンソン病、注意欠陥多動性障害、統合失調症、および薬物中毒）。

これらの環境の操作にさらさ成体マウスは、中脳の数字（SNCとVTA）を変更されたではなく、LC、TH +ニューロン、及びEEプラスピクロトキシンまたはビククリンどちらの同時中脳注入（GABA受容体拮抗薬）は、よりのSNc TH +のEE-誘導を廃止しているニューロン。これらのデータは、以前に6に掲載された。本発明のデータは、その以前の研究の一環として行わ反復実験でコンパイルされた?...

Watch this Scientific Journal Video about Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice at JoVE.com

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

成体マウスにおける中脳ドーパミンニューロンの数の環境モジュレーション

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

environmental-modulations-number-midbrain-dopamine-neurons-adult

Research

JoVE Journal

Neuroscience

8.7K ビュー.  The University of Melbourne. This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

ビデオ: Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

ダイナミッククランプ法を用いたラット中脳ドーパミン作動性ニューロンにおけるNMDA受容体のコンダクタンスのアプリケーション

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

神経科学

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

アダルト生まれのニューロンの統合を検討し

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

不定冠詞<em&gt;インビトロ</em成体マウスの筋層間神経叢から分離腸ニューロンとグリアの&gt;準備

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

グルコース感知を勉強する成体マウス由来の膜視床下部腹内側部のニューロンの電位色素イメージング

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

誘導された神経細胞への成人の脳の周皮細胞由来細胞の系統の再プログラミング

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

レーザーキャプチャーマイクロダイセクション - 個々のドーパミンニューロンの単離と全体の腹側被蓋領域の実証

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

黒質ドーパミンニューロンの細胞体樹状突起のドメインからの亜細胞パッチクランプ記録

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

蛍光プローブを用いた培養ニューロンにおけるエンドソームおよびリソソームの動力学の定量

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

高速液体クロマトグラフィー分析とシナプトソーム ドーパミンによるマウスにおけるドーパミン作動性恒常性の評価

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

さまざまな地域と発展途上と大人の脳のラミナを明らかにするシナプス亜鉛化学の使用

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...