Name: comprehensive profiling of dopamine regulation in substantia nigra and ventral tegmental area
Uploaded: 2012-08-10T13:00:00
Description: Watch this Scientific Journal Video about Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area at JoVE.com

这项工作，并作为引2,10，提供了资金，部分研究给予奖励为MF的Salvatore从美国联邦老龄化研究，爱德华·斯蒂尔斯信托基金和路易斯安那州西北的生物医学研究基金会，艾克Muslow博士前奖学金Pruett学士学位，路易斯安那州立大学健康科学中心什里夫波特。

1。解剖<ol><li>在湿冰床，放松啮齿类动物的大脑基质（冠状切片分离1 mm处），培养皿中含有5剃须刀，＃11手术刀。在一个单独的容器中，地方标记成干冰2毫升大小的离心管。 </li><li>研究者会选择安乐死的方法。我们有可重复性的结果，理想的情况下，异氟醚蒸发器应该被用来进行非常简短的麻醉。但是，如果不提供，我们使用一个大型的电瓶包含一个平台。在位于批准通风罩电瓶，将异氟醚?...

<ol>
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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. 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正如图1中所述，上面详细介绍的方法应该产生多巴胺和调节蛋白TH，的DAT，从一个样品SN或腹侧被盖区VMAT2的大鼠或小鼠获得多个读数。再次，开展这项协议的好处是，研究人员可以得到多巴胺是如何调节体内几乎所有的实验范式下，这样做，保存在任何所需的动物数量减少的重要实验资源的业务相匹配的读数实验。 这是绝对必要的，研究者观察在夹层（4℃），存储组...

高效液相色谱系统： 基本系统包括岛津LC10中ADVP HPLC泵，水WISP的717自动进样器，250×4.4毫米5微米Spherosorb的ODS-1 C18反相柱（水域），生化分析系统（BAS），TL12双玻碳电极，的BAS LC4B电化学探测器和水域授权2数据收集和集成系统。 列保持在30-45℃（BAS LC22A列加热器）。流动相为0.1 M磷酸钠（pH值3.0），0.1 mM的EDTA，1辛烷磺酸（伊斯曼 - 柯达公司）0.2-0.4毫米，0.35％乙腈（V / V），通过0.45微米的过滤器过滤。流速是1.2毫升/分钟。通过调节pH值，辛烷磺酸和柱温，流动相升批次优化分离。回收流动相，并不断与氦气吹扫，以去除水中溶解的氧气。回收的流动相几乎是必不可少的一个合理的时间内保持良好的分辨率。保持流动相保质期，使用流量开关（集成控制）转移到浪费每次运行2-7分钟。 一个Ag/AgC1参比电极的电极保持在约0.78和0.95V的潜力。电极在较高的潜在专门用于测定色氨酸（NMDA受体内部标准）。 0.78 V电位检测的单胺类化合物，色氨酸等，提供了优越的信噪比。色谱存储在硬盘驱动器的授权工作站，和随后的处理和数据直接转入Excel电子表格中代谢物的数额和编制组数据的计算。 泵：岛津LC-10AD 手机：BAS横流。玻碳电极在0.780和0.950 V的潜力。 探测器：BAS的LC-4B在双通道模式运作。 数据ACQ。系统：沃特授权Pro 2的。 喷油器：水域的WISP 717 列：Spherosorb水域消耗臭氧层物质-1，5微米的颗粒，4.4毫米×250毫米。 <table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>十二烷基硫酸钠（SDS） </td><td> - 托德·贝克</td><td> 4095-02 </td></tr><tr><td> trizma基地</td><td>西格玛</td><td> T1503-1KG </td></tr><tr><td> trizma盐酸</td><td>西格玛</td><td> T3253-1KG </td></tr><tr><td>甘油</td><td>西格玛</td><td> G8773-500毫升</td></tr><tr><td> PVP-40 </td><td>西格玛</td><td> PVP40-1KG </td></tr><tr><td> DPBS </td><td> Gibco公司</td><td> 21600-069 </td></tr><tr><td> tween20 </td><td>西格玛</td><td> P1379-500毫升</td></tr><tr><td>甘氨酸</td><td>西格玛</td><td> G8898-1KG</td></tr><tr><td>丽春红S </td><td> Fluka公司</td><td> 81460 </td></tr><tr><td>溴酚蓝</td><td>西格玛</td><td> B8026-5G </td></tr><tr><td>二硫苏糖醇</td><td>西格玛</td><td> D - 9163 </td></tr><tr><td> 2毫克BSA蛋白标准</td><td>西格玛</td><td> P5619-25VL </td></tr><tr><td>皮尔斯BCA蛋白检测试剂A </td><td>热尔科技</td><td> 23223 </td></tr><tr><td>精密加蛋白标准</td><td>生物拉德</td><td> 161-0373 </td></tr><tr><td> [125I]-A蛋白，特定活动</td><td>在Perkin-Elmer </td><td></td></tr></tbody></table> 表2。特定的试剂。 <table border="1"><tbody><tr><td> 试剂 </td><td> 公式 </td></tr><tr><td> 10％SDS </td><td> 10克SDS的100毫升DI H 2 O </td></tr><tr><td> 1％SDS（pH值至8.2） </td><td><ol><li> 10毫升10％SDS </li><li> 60.5毫克Trizma基地</li><li> 37.22毫克EDTA </li><li> 90毫升DI H 2 O </li></ol></td></tr><tr><td>二铜硫酸解</td><td><ol><li> 1 G铜二硫酸</li><li> 25毫升DI H 2 O </li></ol></td></tr><tr><td> 3X样品缓冲液</td><td><ol><li> trizma基地2.27克</li><li> SDS6克</li><li>二硫苏糖醇0.463克</li><li>甘油30克</li><li>溴酚蓝10毫克</li><li>谛2 0（最初加入上述试剂在量筒至40毫升的H 2 0，然后加入H 2 O，直到量达到100毫升） </li><li>盐酸（添加所需的pH值达到6.85） </li><li>冻结在5000 2.0 mL管的解决方案。 </li></ol> （100毫升）：需要的3倍样品缓冲液量=半SDS的样本量。 </td></tr><tr><td> 1X样品缓冲液</td><td>稀释3倍样品缓冲液1X样品缓冲液使用的DI H 2 O </td></tr><tr><td> 10X<html>运行缓冲液（4升） </td><td><ol><li> trizma基地121.1克</li><li>甘氨酸577克</li><li> SDS40克</li></ol></td></tr><tr><td> 10X传输缓冲区：（4升） </td><td><ol><li>甘氨酸360克</li><li> trizma基地96克</li></ol></td></tr><tr><td>丽春</td><td><ol><li>丽春红S。0.5​​克</li><li>乙酸5毫升</li><li> DI H 2 0 95毫升</li></ol></td></tr><tr><td> 0.2％的盐酸溶液</td><td>在500毫升的DI H 2 O 5.2 mL盐酸</td></tr><tr><td> PVP-T20阻断溶液（4升） </td><td><ol><li> PVP的4040克</li><li> DPBS38.2克</li><li> tween202克</li><li> thimerisol0.4克</li><li> 1M的Tris pH值7.6（60.6克三盐酸+13.9克Tris碱在500毫升DI H 2 O） - 50毫升</li></ol></td></tr><tr><td> 10X印迹缓冲（4升） </td><td><ol><li>吐温2020克</li><li> Tris碱14克的</li><li>三盐酸61克的</li></ol></td></tr></tbody></table>内容】“&gt; 表3。蛋白质含量和蛋白质印迹公式。 酪氨酸羟化酶标准：标定的TH蛋白和磷酸化本实验室所使用的标准是来自PC12细胞提取物，对先前的校准TH标准，最终从实验室博士约翰海科克11起源的TH蛋白含量和磷酸化化学计量分析。

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

多巴胺是一种中枢神经系统的大力研究的神经递质。事实上，在自发活动的参与和奖励有关的行为，促进多巴胺调控的分子缺陷五十年的调查。纹状体和伏隔核的多巴胺调控时，大脑的分子基础，其在终端领域的黑质纹状体和mesoaccumbens途径地区的监管重点在这些查询大多数。此外，这些研究都集中在多巴胺的组织内容的分析与正常化的唯一的湿纸巾重量。调查的蛋白质，调节多巴胺，如酪氨酸羟化酶（TH）蛋白，TH磷酸化，多巴胺转运体（DAT），2囊泡单胺转运蛋白（VMAT2），通常不包括多巴胺的组织内容在同一样品的分析。同时分析多巴胺的组织内容及其调节蛋白（包括后TRA的能力nslational修改），不仅提供了内在的力量来解释多巴胺与TH，DAT或VMAT2蛋白水平和功能的关系，而且还扩展了样本经济。这意味着到更低的成本，但会产生多巴胺的分子调控，在几乎所有的研究者选择的范式的见解。 我们的分析集中在中脑。虽然最多巴胺调控的研究通常被忽视的SN和腹侧被盖区，这些原子核很容易解剖与实践。可以进行一个全面的读数，多巴胺的组织内容和TH的DAT，或VMAT2。行为在SN和腹侧被盖区的多巴胺功能的影响有正蓬勃发展的文学，和外源性物质或疾病的进程，其中1-5 impingements。此外，如生长因子的化合物对多巴胺和多巴胺调节蛋白产生深远的影响，到相对更大程度上的SN或腹侧被盖区<sup&gt; 6-8。因此，这种方法提出参考实验室，希望延长其具体的治疗方法如何调节行为和多巴胺调控的查询。在这里，一个多步骤的方法提出了多巴胺的组织内容的分析，的TH蛋白水平的DAT或VMAT2，啮齿动物脑黑质和腹侧被盖区的磷酸化和TH。 TH的磷酸化分析，不仅是如何调节TH活力，而且还影响级联信号在一个给定的范例somatodendritic核可以产生显着的见解。 我们将说明夹层技术来分隔这两个核和解剖组织来样加工，产生的文件，揭示在体内的多巴胺调控的分子机制，具体的每一个细胞核（图1）。

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

多巴胺是明显的调节中脑核，其中包含的多巴胺神经元的胞体和树突。在这里，我们描述了一次解剖和样品处理方法效果发挥到极致，因此，结论和见解，在黑质（SN）和腹侧被盖区（VTA）在啮齿动物的脑核的多巴胺调节。

在黑质和腹侧被盖区多巴胺调控的综合性能分析

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

Research

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单细胞的多巴胺释放，同时电压钳和电流分析法测量

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

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创新 RNA原位检测技术在 D1-Alpha 鼠伏隔核中多巴胺受体的鉴定

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多发性硬化患者综合解剖计划

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

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

联合输注和刺激与快速扫描循环伏安法（CIS-FSCV）评估腹侧被盖区域受体对相位多巴胺的调节

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