Name: environmental modulations of the number of midbrain dopamine neurons in adult mice
Uploaded: 2015-01-20T14:00:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice at JoVE.com

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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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. J., Noonan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+of+laboratory+rats+for+potentially+enriching+stimulus+objects.">Preference of laboratory rats for potentially enriching stimulus objects.</a> Laboratory animals. 30, 97-101 (1996).</li><li>Hanmer, L. A., Riddell, P. M., Williams, 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=Using+a+runway+paradigm+to+assess+the+relative+strength+of+rats'+motivations+for+enrichment+objects.">Using a runway paradigm to assess the relative strength of rats' motivations for enrichment objects.</a> Behavior research methods. 42, 517-524 (2010).</li><li>Burrows, E. L., McOmish, C. E., 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=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. M., Baumans, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strength+of+preference+for+nesting+material+as+environmental+enrichment+for+laboratory+mice.">Strength of preference for nesting material as environmental enrichment for laboratory mice.</a> Applied Animal Behaviour Science. 55, 369-382 (1998).</li><li>Rampon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+environmental+enrichment+on+gene+expression+in+the+brain.">Effects of environmental enrichment on gene expression in the brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 12880-12884 (2000).</li><li>Eckert, M. J., Abraham, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+environmental+enrichment+exposure+on+synaptic+transmission+and+plasticity+in+the+hippocampus.">Effects of environmental enrichment exposure on synaptic transmission and plasticity in the hippocampus.</a> Current topics in behavioral neurosciences. 15, 165-187 (2013).</li><li>Sztainberg, Y., Chen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+environmental+enrichment+model+for+mice.">An environmental enrichment model for mice.</a> Nature protocols. 5, 1535-1539 (2010).</li><li>Sale, A., Berardi, N., Maffei, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environment+and+brain+plasticity:+towards+an+endogenous+pharmacotherapy.">Environment and brain plasticity: towards an endogenous pharmacotherapy.</a> Physiological reviews. 94, 189-234 (2014).</li></ol>

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><tbody><tr><td>Isofluorane</td><td>Baxter Healthcare Pty Ltd, Baxter Drive, NSW 2146, Australia</td><td>AHN3640</td><td></td></tr><tr><td>ALZET Osmotic pump 1002</td><td>DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td><td>0004317</td><td></td></tr><tr><td>ALZET Brain infusion kit 1</td><td>DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td><td>0004760</td><td></td></tr><tr><td>ALZET cannula holder 1</td><td>DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td><td>0008860</td><td></td></tr><tr><td>Vertex Monomer Self-curing (dental acrylic solvent)</td><td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td><td>n/a</td><td></td></tr><tr><td>Vertex Self Curing (dental acrylic powder)</td><td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td><td>n/a</td><td></td></tr><tr><td>METACAM (Meloxicam)</td><td>Troy Laboratories, 98 long Street, smithfield NSW 2164 Australia</td><td>L10100</td><td></td></tr><tr><td>Sodium Pentobarbitone</td><td>Lethabarb, Virbac, Milperra, NSW, Australia</td><td>571177</td><td></td></tr><tr><td>Normal goat serum</td><td>chemicon-temecula, CA</td><td>S26-Litre</td><td></td></tr><tr><td>Triton X-100</td><td>Merck Millipore Headquarters&amp;nbsp;, 290 Concord road, Billerica, MA 01821</td><td>1.08603.1000</td><td></td></tr><tr><td>Polyclonal rabbit anti-tyrosine hydroxylase</td><td>Merck Millipore Headquarters&amp;nbsp;, 290 Concord road, Billerica, MA 01821</td><td>AB152</td><td></td></tr><tr><td>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>EO432</td><td></td></tr><tr><td>Avidin peroxidase</td><td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td><td>A3151-1mg</td><td></td></tr><tr><td>Diamino-benzidine</td><td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td><td>D-5637</td><td></td></tr><tr><td>Stereo Investigator</td><td>MicroBrightField Bioscience, 185 Allen Brook Lane, Suite 101, Williston, VT 05495</td><td>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

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

パーキンソン病のマウスモデルにおける感覚運動機能の評価

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

行動学

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

げっ歯類の新生児から一次分離した中脳ドーパミン細胞培養

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

生物学

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

黒質や腹側被蓋野のドーパミン規制の包括的なプロファイリング

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

神経科学

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

ビジュアル欠陥を特徴づけるために慢性高眼圧症のレーザー誘起マウスモデル

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

医学

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

パーキンソン病モデルマウス：6 - OHドーパミンの病変から行動試験に

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

パーキンソン病における感受性メカニズムのマスク解除を行う遺伝子環境相互作用モデル

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

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 ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

NF-κB依存性神経新生の調節および分析のための方法

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

マウスドーパミン作動性ニューロンの初代培養

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

胚性齧歯類の脳から単離、プライマリ脳ドーパミンニューロンの文化と長期メンテナンス

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...