この作品、として2,10を挙げて 、のための資金は、老化研究、エドワード·P.スタイルズ信託基金とノースウエストルイジアナ州の医学研究財団のためのアメリカ連合からMFサルバトーレの研究助成賞を受賞することによって、部分的には、提供されていましたとBSプルエットにアイクMuslow博士号を取得する前のフェローシップ、LSUの健康科学センターシュリーブポートから。

<ol>
	<li>Trevitt, J. T., Carlson, B. B., Nowend, K., Salamone, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substantia+nigra+pars+reticulate+is+a+highly+potent+site+of+action+for+the+behavioral+effects+of+the+D1+antagonist+SCH23390+in+rat.">Substantia nigra pars reticulate is a highly potent site of action for the behavioral effects of the D1 antagonist SCH23390 in rat.</a> Psychopharmacology. 156, 32-41 (2001).</li><li>Salvatore, M. F., Pruett, B. S., Spann, S. L., Dempsey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+reveals+a+role+for+nigral+tyrosine+hydroxylase+ser31+phosphorylation+in+locomotor+activity+generation.">Aging reveals a role for nigral tyrosine hydroxylase ser31 phosphorylation in locomotor activity generation.</a> PLoS ONE. 4, e8466 (2009).</li><li>Rossato, J. L., Bevilaqua, L. R. M., Izquierdo, I., Medina, J. H., Cammarota, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+controls+persistence+of+long-term+memory+storage.">Dopamine controls persistence of long-term memory storage.</a> Science. 325, 1017-1020 (2009).</li><li>Keller, C. M., Salvatore, M. F., Pruett, B. S., Guerdin, G. F., Goeders, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biphasic+dopamine+regulation+in+mesoaccumbens+pathway+in+response+to+non-contigent+binge+and+escalating+methamphetamine+regimens+in+the+Wistar+rat.">Biphasic dopamine regulation in mesoaccumbens pathway in response to non-contigent binge and escalating methamphetamine regimens in the Wistar rat.</a> Psychopharmacology. 215, 513-526 (2011).</li><li>Lu, L., Dempsey, J., Liu, S. Y., Bossert, J. M., Shaham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+infusion+of+brain-derived+neurotrophic+factor+into+the+ventral+tegmental+area+induces+long-lasting+potentiation+of+cocaine+seeking+after+withdrawal.">A single infusion of brain-derived neurotrophic factor into the ventral tegmental area induces long-lasting potentiation of cocaine seeking after withdrawal.</a> J. Neurosci. 24, 1604-1611 (2004).</li><li>Hoffer, B. J., Hoffman, A., Bowenkamp, K., Huettl, P., Hudson, J., Martin, D., Lin, L. F., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+line-derived+neurotrophic+factor+reverses+toxin-induced+injury+to+midbrain+dopaminergic+neurons+in+vivo.">Glial cell line-derived neurotrophic factor reverses toxin-induced injury to midbrain dopaminergic neurons in vivo.</a> Neurosci. Lett. 182, 107-111 (1994).</li><li>Salvatore, M. F., Zhang, J. L., Large, D. M., Wilson, P. E., Gash, C. R., Thomas, T. C., Haycock, J. W., Bing, G., Stanford, J. A., Gash, D. M., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Striatal+GDNF+administration+increases+tyrosine+hydroxylase+phosphorylation+in+the+rat+striatum+and+substantia+nigra.">Striatal GDNF administration increases tyrosine hydroxylase phosphorylation in the rat striatum and substantia nigra.</a> J. Neurochem. 90, 245-254 (2004).</li><li>Lu, L., Wang, X., Wu, P., Xu, C., Zhao, M., Morales, M., Harvey, B. K., Hoffer, B. J., Shaham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ventral+tegmental+area+glial+cell-line+derived+neurotrophic+factor+in+incubation+of+cocaine+craving.">Role of ventral tegmental area glial cell-line derived neurotrophic factor in incubation of cocaine craving.</a> Biol. Psychiatry. 66, 137-145 (2009).</li><li>Lavicky, J., Dunn, 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=Corticotropin-releasing+factor+stimulates+catecholamine+release+in+hypothalamus+and+prefrontal+cortex+in+freely+moving+rats+as+assessed+by+microdialysis.">Corticotropin-releasing factor stimulates catecholamine release in hypothalamus and prefrontal cortex in freely moving rats as assessed by microdialysis.</a> J. Neurochem. 60, 602-612 (1993).</li><li>Salvatore, M. F., Pruett, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dichotomy+of+tyrosine+hydroxylase+and+dopamine+regulation+between+somatodendritic+and+terminal+field+areas+of+nigrostriatal+and+mesoaccumbens+pathways.">Dichotomy of tyrosine hydroxylase and dopamine regulation between somatodendritic and terminal field areas of nigrostriatal and mesoaccumbens pathways.</a> PLoS ONE. 7, e29867 (2012).</li><li>Salvatore, M. F., Garcia-Espana, A., Goldstein, M., Deutch, A. Y., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+tyrosine+hydroxylase+phosphorylation+in+the+nigrostriatal+and+mesolimbic+systems+in+vivo:+Effects+of+acute+haloperidol+and+related+compounds.">Stoichiometry of tyrosine hydroxylase phosphorylation in the nigrostriatal and mesolimbic systems in vivo: Effects of acute haloperidol and related compounds.</a> J. Neurochem. 75, 225-232 (2000).</li><li>Salvatore, M. F., Waymire, J. C., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depolarization-stimulated+catecholamine+biosynthesis:+involvement+of+protein+kinases+and+tyrosine+hydroxylase+phosphorylation+sites+in+situ.">Depolarization-stimulated catecholamine biosynthesis: involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ.</a> J. Neurochem. 79, 349-360 (2001).</li><li>Haycock, J. W., Lew, J. Y., Garcia-Espana, A., Lee, K. Y., Harada, K., Meller, E., Goldstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+serine-19+phosphorylation+in+regulating+tyrosine+hydroxylase+studied+with+site-+and+phosphospecific+antibodies+and+site-directed+mutagenesis.">Role of serine-19 phosphorylation in regulating tyrosine hydroxylase studied with site- and phosphospecific antibodies and site-directed mutagenesis.</a> J. Neurochem. 71, 1670-1675 (1998).</li><li>Lindgren, N., Xu, Z. Q., Linskog, M., Herrera-Marschitz, M., Goiny, M., Haycock, J. W., Goldstein, M., Hokfelt, T., Fisone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+tyrosine+hydroxylase+activity+and+phosphorylation+at+ser19+and+ser40+via+activation+of+glutamate+NMDA+receptors+in+rat+striatum.">Regulation of tyrosine hydroxylase activity and phosphorylation at ser19 and ser40 via activation of glutamate NMDA receptors in rat striatum.</a> J. Neurochem. 74, 2470-2477 (2000).</li></ol>

利害の衝突が宣言されません。

図1に概説され、上記詳述した方法は、ドーパミンの複数の読み出しおよびラットまたはマウスから得られたSNまたはVTAの1つのサンプルから、その調節タンパク質TH、DAT、およびVMA​​T2を生成する必要があります。再び、このプロトコルを実施する利点は、研究者はドーパミンが実質的にすべての実験的なパラダイムの下でin vivoでの規制と、そうすることで、いずれかに必要な動物数?...

HPLCシステム： 基本的なシステムは島津LC10-ADvp HPLCポンプ、ウォーターズWISP 717自動サンプルインジェクター、250 X 4.4ミリメートル、5ミクロンSpherosorb ODS-1 C18逆相カラム（Waters）、バイオ分析システム（BAS）TL12デュアルガラスで構成されています炭素電極、2 BAS LC4B電気化学検出器、およびウォーターズは、2のデータ収集およびデータの統合システムに力を与える。 列は30から45°C（BAS LC22Aカラムヒーター）に維持されています。移動相は0.1 Mリン酸ナトリウム液（pH3.0）、0.1mMのEDTA、0.2から0.4 mMの1 - オクタンスルホン酸（イーストマンコダック - ）、および0.35％0.45ミクロンフィルターを通して濾過し、アセトニトリル（v / v）である。流量は1.2 ml /分である。移動相の4リットルのバッチは、pH、オクタンスルホン酸およびカラム温度を調整することにより、分離のために最適化されています。移動相はリサイクルされており、継続的に溶存酸素を除去するためにヘリウムガスでパージされています。移動相のリサイクル合理的な期間のために良好な分解能を維持するためにほとんど不可欠である。移動相の貯蔵寿命は、各実行の最初の2から7分を無駄にそらすために、フロースイッチ（積分器によって制御される）を使用することによって維持されています。 電極はAg/AgC1参照電極に対して約0.78と0.95Vの電位で維持されています。より高い電位での電極は、トリプトファン（およびNMDA内部標準）を決定するために排他的に使用されます。 0.78 Vの電位はトリプトファン以外のモノアミン及びその化合物の検出のための雑音比に優れた信号を提供します。クロマトグラムをエンパワーワークステーションのハードドライブに保存され、その後、処理され、データは代謝産物の量およびグループのデータのコンパイルを計算するためのExcelスプレッドシートに直接転送されます。 ポンプ：島津LC-10AD 細胞：BASクロスフロー。 0.780および0.9で作用電極グラッシーカーボン50 Vの可能性。 検出器：BAS LC-4Bは、デュアルチャネルモードで動作する。 データACQ。システム：ウォーターズはPro 2をエンパワー。 インジェクター：ウォーターズWISP 717 カラム：ウォーターズSpherosorb ODS-1、5μm粒子、4.4ミリメートルX 250ミリメートル。 <table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>ドデシル硫酸ナトリウム（SDS） </td><td> - JTベーカー</td><td> 4095から02 </td></tr><tr><td> Trizmaベース</td><td>シグマ</td><td> T1503-1KG </td></tr><tr><td> Trizma HClを</td><td>シグマ</td><td> T3253-1KG </td></tr><tr><td>グリセロール</td><td>シグマ</td><td> G8773-500 mLの</td></tr><tr><td> PVP-40 </td><td>シグマ</td><td> PVP40-1KG </td></tr><tr><td> DPBS </td><td>ギブコ</td><td> 21600-069 </td></tr><tr><td> Tween20を</td><td>シグマ</td><td> P1379-500 mLの</td></tr><tr><td>グリシン</td><td>シグマ</td><td> G8898-1KG</td></tr><tr><td>ポンソーS </td><td>フルカ</td><td> 81460 </td></tr><tr><td>ブロモフェノールブルー</td><td>シグマ</td><td> B8026-5G </td></tr><tr><td>ジチオスレイトール</td><td>シグマ</td><td> D-9163 </td></tr><tr><td>タンパク質標準2mgのBSA </td><td>シグマ</td><td> P5619-25VL </td></tr><tr><td>ピアスBCAタンパク質アッセイ試薬</td><td>サーモフィッシャーサイエンティフィック</td><td> 23223 </td></tr><tr><td>プレシジョンプラスプロテインスタンダード</td><td>バイオラッド社</td><td> 161-0373 </td></tr><tr><td> [125] - 蛋白質、特定のアクティビティ</td><td>パーキンエルマー</td><td></td></tr></tbody></table> 表2特定の試薬 ​​が含まれています。 <table border="1"><tbody><tr><td> 試薬 </td><td> 式 </td></tr><tr><td> 10％SDS </td><td> 10グラムSDS、100 mLのDI H 2 0 </td></tr><tr><td> 1％SDS液（pH〜8.2） </td><td><ol><li> 10 mLの10％SDS </li><li> 60.5 mgのTrizmaベース</li><li> 37.22 mgのEDTA </li><li> 90mLのDI H 2 0 </li></ol></td></tr><tr><td>銅II硫酸溶液</td><td><ol><li> 1グラムの銅II硫酸</li><li> 25mLのDI H 2 0 </li></ol></td></tr><tr><td> 3Xサンプルバッファー</td><td><ol><li> Trizmaベース2.27グラム</li><li> SDS 6グラム</li><li>ジチオスレイトール0.463グラム</li><li>グリセロール30グラム</li><li>ブロモフェノールブルー10mgを</li><li> DIH 2 0（最初にメスシリンダーにH 2 O 40 mLに上記の試薬 ​​を加える;体積を100 mLに達するまで、その後H 2 0を追加） </li><li>塩酸（6.85のpHに到達するために、必要に応じて追加） </li><li> 50 2.0 mLチューブ内の溶液を凍結する。 </li></ol> （100 mLを行う）：3Xサンプルバッファーのボリュームは、サンプルで使用されているSDSの=½のボリュームを必要としていました。 </td></tr><tr><td> 1Xサンプルバッファー</td><td> DI H 2 0を使用して1Xサンプルバッファーに3Xサンプルバッファーを希釈する</td></tr><tr><td> 10X<html>ランニング緩衝液（4 Lになります） </td><td><ol><li> Trizmaベース121.1グラム</li><li>グリシン577グラム</li><li> SDS 40グラム</li></ol></td></tr><tr><td> 10X転送バッファ（4 Lになります） </td><td><ol><li>グリシン360グラム</li><li> Trizmaベース96グラム</li></ol></td></tr><tr><td>ポンソー</td><td><ol><li>ポンソーS·0.5グラム</li><li>酢酸5 mLを</li><li> DI H 2 0〜95 mLの</li></ol></td></tr><tr><td> 0.2パーセントのHCl溶液</td><td> DI H 2 O 500mlを5.2 mLの塩酸</td></tr><tr><td> PVP-T20はSolnをブロックします。 （4 Lになります） </td><td><ol><li> PVP-40 40グラム</li><li> DPBS 38.2グラム</li><li> Tween20を2グラム</li><li> Thimerisol 0.4グラム</li><li> 1MトリスpHは7.6（60.6グラムトリスHCl + 500mlのDI H 2 0 13.9グラムトリスベース） - 50 mLの</li></ol></td></tr><tr><td> 10Xブロット緩衝液（4 Lになります） </td><td><ol><li> Tween 20を20グラム</li><li>トリスベース14グラム</li><li>トリスHCl 61グラム</li></ol></td></tr></tbody></table>ontent &quot;&gt; 表3。タンパク質アッセイおよびウェスタンブロッティング式。 チロシン水酸化酵素の基準：この研究室で使用されるキャリブレーションTH蛋白質およびリンの基準は、最終的にドクター·ジョン干し草の山11の研究室に由来する以前にキャリブレーションTH規格に対してTHタンパク質含有量とリン化学量論を分析したPC12細胞抽出物から誘導される。

comprehensive profiling of dopamine regulation in substantia nigra and ventral tegmental area

ドーパミンは中枢神経系で精力的に研究神経伝達物質である。確かに、その自発運動への関与と報酬に関連する動作は、ドーパミン調節に関連する分子の不備にお問い合わせの五十年を促進しています。線条体および側坐核、黒質線条体とmesoaccumbens経路のターミナルフィールド領域におけるその調節の分子基盤に応じて脳フォーカスのドーパミン調節のこれらの問い合わせが多数。さらに、このような研究が唯一のウェットティッシュの重量に正規化とドーパミン組織含有量の分析に集中している。そのようなチロシンヒドロキシラーゼ（TH）タンパク質、THのリン酸化、ドーパミントランスポーター（DAT）、および小胞モノアミン輸送体2（VMAT2）タンパク質として、ドーパミン調節するタンパク質の研究は、しばしば同一のサンプルにおけるドーパミン組織含有量の分析が含まれていません。ドーパミン組織のコンテンツとその調節タンパク質（ポスト-TRAを含む両方を分析する能力nslational変更）TH、DATのタンパク質レベルと機能とドーパミンの関係を解釈する固有の力を与える、またはVMAT2だけでなく、サンプルの経済を拡張するだけでなく。この低コストに変換し、さらには研究者の選択の事実上すべてのパラダイムにおけるドーパミンの分子制御への洞察を生成します。 我々は中脳での分析の焦点を合わせる。 SNとVTAは、通常、ドーパミン調節のほとんどの研究では無視されていますが、これらの核は簡単に実践して解剖されています。包括的なドーパミン組織のコンテンツとTHの読み出し、DAT、またはVMAT2を行うことができる。そこに行動上のSNとVTAのドーパミン機能の影響に関する文献を急成長し、そこに1から5外因性物質または疾患過程のimpingementsされています。さらに、成長因子などの化合物は、SNまたはVTAで比較的大きい程度に、ドーパミンやドーパミン調節蛋白質に大きな影響を持っている<sup&gt; 6-8したがって、この方法論は、特定の治療法は行動とドーパミンの調節を調節する方法でそれらの照会を拡張する研究室への参照のために提示されています。ここでは、マルチステップ法がげっ歯類の脳から黒質およびVTAドーパミン組織からコンテンツの分析、THのタンパク質レベル、DAT、またはVMAT2、およびTHリン酸化のために提示されています。 THリン酸化の解析だけではなく、TH活性が調節されている方法ではなく、与えられたパラダイムの細胞体核に影響を受けたシグナル伝達カスケードに重要な洞察を得ることができます。 我々は、これらの2つの核とそれぞれの原子核（図1）の特定のin vivoでのドーパミン調節の分子機構を明らかにプロファイルを生成し、解剖組織サンプル処理を分離するために解剖の手法を説明します。

Watch this Scientific Journal Video about Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area at JoVE.com

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

ドーパミンは、はっきりとドーパミンニューロンの細胞体と樹状突起を含む脳核、で規制されています。ここでは黒質（SN）とげっ歯類では腹側被蓋野（VTA）の中​​脳核のドーパミン調節では、結果を最大化するために解剖し、サンプル処理のアプローチを説明し、その結果、結論と洞察力。

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 ...

comprehensive-profiling-dopamine-regulation-substantia-nigra-ventral

Research

JoVE Journal

Neuroscience

25.6K ビュー.  Louisiana State University Health Sciences Center. ドーパミンは、はっきりとドーパミンニューロンの細胞体と樹状突起を含む脳核、で規制されています。ここでは黒質（SN）とげっ歯類では腹側被蓋野（VTA）の中​​脳核のドーパミン調節では、結果を最大化するために解剖し、サンプル処理のアプローチを説明し、その結果、結論と洞察力。

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

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

同時に電圧クランプとアンペロとドーパミン放出の単一細胞計測

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

神経科学

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

腹チック中脳マイクロRNAの発現OVO中

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

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

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

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, ...

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 ...

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) ...

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

光分留と標準的な顕微鏡装置を用いたマウス中脳のドーパミン作動性ニューロン数の薬剤の評価

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

検出技術その場で革新的な RNA 側坐齧歯動物の核内ドーパミン D1-アルファ受容体の同定

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

膠様質ニューロンの細胞パッチク ランプ記録のため急性脊髄スライスの準備

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

多発性硬化症の個人のための包括的な解剖プログラム

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement learning. Understanding how the diverse neuronal inputs into the VTA control phasic DA release can provide a better picture of the circuitry that controls reward processing and reinforcement learning. Here, we describe a method that combines intra-VTA cannula infusions of pharmacological agonists and antagonists with stimulation-evoked phasic DA release (combined infusion and stimulation, or CIS) as measured by in vivo fast-scan cyclic voltammetry (FSCV). Using CIS-FSCV in anesthetized rats, a phasic DA response can be evoked by electrically stimulating the VTA with a bipolar electrode fitted with a cannula while recording in the nucleus accumbens core. Pharmacological agonists or antagonists can be infused directly at the stimulation site to investigate specific VTA receptors&#39; roles in driving phasic DA release. A major benefit of CIS-FSCV is that VTA receptor function can be studied in vivo, building on in vitro studies.

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

The goal of this protocol is to directly manipulate ventral tegmental area receptors to study their contribution to subsecond dopamine release.

高速スキャン環状ボルタンメトリー(CIS-FSCV)による注入と刺激を組み合わせて、パジカルドーパミンの腹側テグメンタル領域受容体調節を評価する

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement ...