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，将异氟醚饱和的纱布下面的平台，并等待3-5分钟，以使异氟醚饱和的电瓶。将里面的老鼠和老鼠已经失去了知觉，尽快消除斩首。迅速取出大脑（最好是在两分钟内），冷水下冲洗，并立即放置在冷冻啮齿动物脑矩阵。 </li><li>一分钟后的位置ING矩阵大脑，冷刀片，插入冷却大脑纹状体和伏隔核，如果除了中脑核分析所需的约2毫米，喙视交叉开始的。继续蹒跚其后每刀片的位置，以便更容易去除每一个人的刀片插入寒冷的刀片，每个部分通过桥，直到抵达中途1毫米。 </li><li>当拉出剃须刀，海马开始寻找。当它已经完全包裹周围脑，开始采取部分使用11＃手术刀刀片， 如图2所示。 </li><li>立即放入预冷的离心管干冰解剖组织。在大鼠，期望能够从两个冠状片的最低和最高的三个解剖SN和腹侧被盖区。商店组织于-80°C，直到准备处理。 </li></ol> 2。组织分析：高效液相色谱法f或多巴胺和代谢产物高效液相色谱设备用品及维修的完整信息，请参阅高效液相色谱分析，在手稿年底。 <ol><li>轻轻地从干冰使用冰冷的0.1 M的高氯酸4 - EDTA溶液（0.1 M的高氯酸及0.1 mM的EDTA），其中包含在N-甲基多巴胺浓度20毫微克/毫升，作为中取出后，立即组织匀浆高效液相色谱分析样品的复苏过程中的内部标准。我们使用的是均质1布兰森Sonifier 150，在全功率的10％（10 1）设置。以下的超声卷的建议： 在大鼠，SN = 250μL（单方面夹层），400μL（双侧夹层），腹侧被盖区= 200μL（单方面夹层），400μL（双边夹层）。 在鼠标，（只有双边夹层），SN = 200μL，腹侧被盖区= 200μL。 </li><li>同质化后，干冰样品。样品然后centrifuged 12,000 RPM（杜邦Sorvall Microspin 24S）的蛋白质沉淀物颗粒可用于免疫印迹测定。上清直接注射到高效液相色谱法。以下是建议的注射量在大鼠或小鼠精确的分析：（无论从准备）SN = 150（或者准备），腹侧被盖区= 160μL </li><li>股票的标准准备在1毫克/毫升，加入适量的单胺类神经递质及代谢产物的多巴胺成分0.1米的高氯酸0.1 mM的EDTA。每个化合物的浓度为1毫克/毫升，除色氨酸是5毫克/毫升。股票的标准分为10μL分装，储存在超低直到需要。 *所有的重量，如果适用的复合盐的形式纠正。 准备100毫升的高氯酸溶液稀释10μL等份的股票标准的工作标准。工作标准的浓度为0.1微克/毫升，除色氨酸为0.5微克/毫升。 50μL的这方面的工作标准，注入高效液相色谱法，除色氨酸是25纳克的每个组件的5毫微克列金额。 </li><li>多巴胺回收样本等份内的测定： 有几个步骤，涉及到确定从样品等分多巴胺恢复。 </li><li>首先，除以峰高标准的多巴胺多巴胺样品的峰高和这个比例乘以总金额多巴胺（纳克）的标准。我们标准的多巴胺浓度为0.1纳克/μL和50μL标准检测。因此，标准中的多巴胺数量为5毫微克。因此，下面的等式可以用来确定一个给定样品中的多巴胺恢复： （多巴胺标准样品/多巴胺峰高峰高）* 5纳克</li><li>校正样品使用内部标准的N-甲基多巴胺的损失： 实际恢复的N-甲基多巴胺多巴胺同样的方式计算，除以N-甲基DOP胺N-甲基多巴胺的标准峰的高度和这个比例乘以总金额N-甲基多巴胺（NG）的标准样品的峰高。多巴胺一样，我们的标准的N-甲基多巴胺浓度为0.1 ng /μL。 50μL的标准检测。因此，标准的N-甲基多巴胺金额为5毫微克。因此，下面的等式可以用来确定一个给定的样品中N-甲基多巴胺恢复： （N-甲基多巴胺样品的峰高/ N-甲基多巴胺标准峰高）* 5ng 然而，由于不是从每个样品中的N-甲基多巴胺样品本身，而是从HPLC缓冲区，预计在每个样品的N-甲基多巴胺金额计算。在高效液相色谱法缓冲N-甲基多巴胺浓度为20 ng / mL或0.02毫微克/μL，所以N-甲基多巴胺在每个样品的预期金额是由等分量乘以N-甲基多巴胺浓度的高效液相色谱缓冲发现每个样品为HPLC分析。这表现在以下公式： （0.02毫微克/μL）*（等分样品量的高效液相色谱分析） </li><li>预期和恢复的N-甲基多巴胺之间的差异可以用来纠正任何样品损失调整为多巴胺回收金额。简单地采取比预期的N-甲基多巴胺/恢复N-甲基多巴胺和多巴胺的回收量乘以。因此，样品中的等份的金额为多巴胺的整体方程如下： （多巴胺标准样品/多巴胺峰高峰高）*（N-甲基多巴胺标准/ N-甲基多巴胺样品的峰高峰高）*（0.02毫微克/μL）*（等分样本量用于高效液相色谱分析） </li><li>共有多巴胺回收样品： 从样品等分恢复多巴胺的值，然后用在一个给定的样本来推断多巴胺总额。这是除以回收量（纳克）Of伤残等分体积和样品超声英寸的高效液相色谱法缓冲总额乘以这个值，这是由下面的等式： （取样品中量的多巴胺（NG）/体积样品分装）*（高效液相色谱缓冲区的总成交量到其中的样品，超声）。 读者可以参考以下的结合不仅SN和腹侧被盖区的多巴胺调节的蛋白质，但纹状体和伏隔核以及读数多巴胺及其代谢产物在评估全面介绍研究2,4,10。 </li></ol> 3。组织分析：总蛋白和最终正常化TH和DAT结果<ol><li>放入湿冰沉淀的蛋白质颗粒（来自高效液相色谱法缓冲处理），以维持4°C确保所有缓冲区已被删除，存档（如果需要验证分析）。如果SN或腹侧被盖区是处理双边（只大鼠），添加300微升（SN）或200μL（VTA）的1％的SDS含5毫米的Tris（pH值8.3）和1 mM EDTA的沉淀和超声粉碎。 SN颗粒或100μL加入150μLSDS溶液腹侧被盖区微丸，如果处理从单方面解剖组织。 </li><li> 〜5分钟即可完成蛋白质变性，使冷却至室温的样品将在不久的开水。在这一步确保帽安全举行。 </li><li>确定蛋白质浓度的样品使用BCA分析，包括蛋白质标准曲线（白蛋白标准）范围为2 - 40微克蛋白。检测5μl样品一式三份的数量和使用中值对标准曲线，以确定蛋白质的数量。除以蛋白浓度的检测量。注意：这是第一正常化总TH，DAT和VMAT2结果总蛋白恢复采取两个步骤。 </li><li>准备减少10％的丙烯酰胺ĞSDS电泳样品缓冲液的样品（含二硫）埃尔斯。理想的情况下，最后的总蛋白浓度应该〜2微克/微升。此浓度的选择的原因是为了减少最终确定的TH磷酸化所必需的样本量，加载精确量化TH磷酸化ser31和ser40〜100μg总蛋白将是必要。如果黄色后加入样品缓冲液样品中，样品是太酸。加入1 M在10μL增量的Tris（pH值8.2），直到恢复蓝色。 </li><li>准备样品为多巴胺调节蛋白的下列裁决如下： </li><li> TH：反对的TH蛋白的校准标准（吴）在SN（纳克每μg蛋白总量TH），TH的浓度预计从约介于0.05-0.27纳克每微克总蛋白2,4次， 7,10,11。反对线性TH标准曲线，范围从0.5 - 5.0纳克TH和使用主要抗体的TH（Millipore公司猫＃AB152，1:1000稀释在PVPţ-20阻断剂），这将产生一个线性的标准曲线，应该从SN总蛋白的最佳负载〜10微克。腹侧被盖区的TH浓度范围从0.4到1.0％μg蛋白4,11吴次。因此，腹侧被盖区的总蛋白负载应该是〜5微克。 </li><li> 的DAT：SN或腹侧被盖区的DAT相对数量明显少于同源终端纹状体和伏隔核4场地区。 TH蛋白恢复正常化的DAT的相对表达也明显在SN和腹侧被盖区。因此，量化SN或腹侧被盖区DAT蛋白的蛋白质负荷需要终端领域的区域样本负载相比要大得多。至少30μg总蛋白的需要精确评估在腹侧被盖区的DAT和额定负载的50μg总蛋白是在SN评估的需要。使用的主要抗体是圣克鲁斯，猫＃SC-1433。 </li><li> VMAT2：VMAT2的表达，作为规范化的总蛋白，是在比黑质纹状体区域mesoaccumbens更大。然而，相对于TH的表达，VMAT2的表达是最伟大的SN和至少在纹状体4。使用圣克鲁斯猫SC-15314＃，标称总蛋白负载的〜40微克，是最好VMAT2量化。 </li><li>运行适当数量的总蛋白的10％丙烯酰胺凝胶上使用的SDS-PAGE。 Bio-Rad公司PROTEAN II电泳装置，用于确定，这是一个大的凝胶格式。蛋白质转移到0.45微米孔径硝化棉。为转移，Hoeffer坦克设置为至少27伏特，允许转移到接管夜间进行。 </li><li>允许的印迹干燥至少30分钟，然后染色与丽春红S溶液（1％，5％冰醋酸丽春红S）〜5分钟。德用0.2％盐酸溶液染色，大力直到个别蛋白带明确解决和背景染色被删除。获得丽春氏染色的形象ING基于Image J在每个车道的相对蛋白负载进一步量化和规范化TH，DAT或VMAT2结果（ 图3）。 </li></ol> 4。组织分析：网站特定TH磷酸在ser31或ser40磷酸的TH可以增加12儿茶酚胺细胞L-多巴的合成。虽然在每个站点的数量要增加L-多巴的合成多巴胺的大脑区域如SN和腹侧被盖区是不成立的磷酸化，有证据表明，ser31磷酸化起着调节L-多巴合成10和DA合作不同的重要作用组织之间的纹状体的内容，SN，伏隔核和腹侧被盖区，10。尽管如此，列入ser40决定是必要的，因为许多研究表明，它可以影响TH的活性，磷酸化的门槛，尤其是如果达到12。 而ser19 phosphorylati不影响TH活动13,14，它可以被视为一个定点的Ca 2 +依赖的多巴胺神经元的信号，由于细胞外Ca 2 +去极化条件下12下增加ser19磷酸化是必要的。此外，还有是一个重大的ser19与ser31只有在SN和腹侧被盖区10的磷酸化的磷酸化的正相关关系，标志着ser19磷酸化状态可能影响ser31磷酸化，从而间接L-多巴合成。 站点特定的TH磷酸化化学计量学的最佳量化的样品负载的考虑：下面，准确和精确的测定特定地点的解剖组织中日磷酸化所需的注意事项。在可能的情况下，校准TH磷酸化标准应包括在样品TH磷酸化的评估。样品负荷必须考虑到ACC使每个磷酸化位点的指望。2-15固有的化学计量学法量化正在使用的抗体的动态工作范围内的磷酸化。作为最后一个音符，固有的总蛋白，每个样品的检测应还装载在标准曲线车道。这很容易实现与载体蛋白（如鼠肝）不表达TH。 <ol><li>的三磷酸化位点。ser19，ser19磷酸化的化学计量水平是最大的SN和腹侧被盖区，范围从0.15 - 0.25 2,10,11。其他的考虑是，通过我们的检测方法，ser19 PS量化的线性范围介于0.5和5.0毫微克磷酸化ser19 10。鉴于这些考虑，总的TH蛋白负载5至10毫微克（从而估计的0.75 - 2.5纳克磷酸化ser19从样品），将提供一个可靠的ser19次磷酸措施。我们使用的主要Phosphosolutions（货号＃p1580-19）1:1000稀释。 </li><li> ser31 ser31磷酸化化学计量学水平是在SN和腹侧被盖区相比，其同源终端领域的区域，范围从0.04 - 0.10 2,4,10,11较少。 ser31 PS我们的检测方法，检测线性范围介于0.5和7.0毫微克磷酸化ser31 10。鉴于这些因素，10和30毫微克之间总的TH蛋白负载（从而估计0.50 - 3.0纳克从样品的磷酸化ser31）将提供可靠的ser31TH磷酸化的措施。参见图4A为的ser31量化的例子。我们的主要抗体是一个内部的亲和纯化抗体制造21世纪生化（马萨诸塞州波士顿），磷酸化表位的特异性验证使用我们的内部标准2。 </li><li> ser40 ser40磷酸化化学计量学水平- SN和腹侧被盖区范围从〜0.02。0.06 2,4,10,11。通过我们的检测方法，SPS ER40检测线性范围为0.2和2.0毫微克磷酸化ser40 10。鉴于这些因素，10和30毫微克之间总的TH蛋白负载（从而估计0.20 - 1.8纳克的样品磷酸化ser40）将提供可靠的ser40TH磷酸化的措施。参见图4B为的ser40量化的例子。我们使用的主要Phosphosolutions（货号＃p1580-40）1:1000稀释。 </li></ol> 5。代表结果<ol><li> SN与腹侧被盖区的多巴胺有三个读数多巴胺（或代谢物）可采取定量分析;为总清扫回收，每蛋白，每TH蛋白（纹状体，SN，伏隔核表明，腹侧被盖在下面的表1）。共有SN和腹侧被盖区多巴胺恢复是相当的可比性，6-9两到三个冠状切片解剖2纳克之间不等。多巴胺恢复正常化的总蛋白，表明THA受雇于吨，根据解剖方法，腹侧被盖区将有更大的多巴胺，平均每人总蛋白6-8 ng / mg蛋白在SN和9-10毫微克/毫克蛋白质在腹侧被盖区，4。最后，多巴胺总TH恢复正常化，还应该揭示了SN和腹侧被盖区之间的差异，在腹侧被盖区10，0.2和0.8之间不等，吴多巴胺每毫微克之间的两个地区2,10日蛋白。 <table border="1"><tbody><tr><td> 多巴胺地区 </td><td> 共有伤残康复 </td><td> 多巴胺每毫克蛋白 </td><td> 多巴胺每纳克TH </td></tr><tr><td> 纹状体 </td><td> 214±16 </td><td> 165±13 </td><td> 0.56±0.11 </td></tr><tr><td> 的SN </td><td> 8.4±0.8 </td><td> 6.1±0.5 </td><td> 0.18±0.03 </td></tr><tr><td> 核交流cumbens </td><td> 60±6 </td><td> 75±4 </td><td> 0.77±0.03 </td></tr><tr><td> 腹侧被盖区 </td><td> 6.0±1.0 </td><td> 9.2±1.4 </td><td> 0.22±0.03 </td></tr></tbody></table></li><li> 总蛋白：TH，DAT，VMAT2使用校准的TH蛋白标准量化，更多的TH恢复SN比从解剖的腹侧被盖区（60纳克在SN和26纳克在腹侧被盖区2）。然而，时归蛋白质，这种关系颠倒，每在腹侧被盖区〜3总蛋白TH - 5倍，比在SN（〜0.32纳克/μg蛋白在VTA和在SN〜0.09）4， 10。 SN和腹侧被盖区的DAT复苏的预期是相似的，稍微恢复在腹侧被盖区为总蛋白或为总TH恢复4。值得注意的是，相比要少得多，在这些地区丰富的DAT蛋白是同源终端领域的地区4 </SUP&gt;。 VMAT2为总蛋白的恢复是在腹侧被盖区与SN 4。然而，当标准化的TH蛋白，有恢复在SN 4稍大。 </li><li> 特定网站从多项研究TH磷酸化 ，有一致的结果，在ser19，ser31，ser40磷酸化在SN和腹侧被盖区的数量，也关系同源终端领域的区域。 ser19磷酸化，特别是在SN和腹侧被盖区（0.2-0.3）相比，纹状体和伏隔核（0.05-0.15）2,4,7,10,11。 ser31磷酸化是明显在SN和腹侧被盖区相比，其同源终端领域的地区，这的2,4,7,10,11 somatodendritic地区之间〜0.06 - 0.10。抑制芳香酸脱羧酶国安局-1015期间，在ser31磷酸化增加2倍，在腹侧被盖区只有10。在SN 0.02和0.05，ser40磷酸范围VTA和一般这种化学计量学不显著不同于同源终端领域的地区2,4,7,10,11。 </li></ol><img alt="图1" src="/files/ftp_upload/4171/4171fig1.jpg" /> 图1。多巴胺读数从一个组织剥离。 <img alt="图2" src="/files/ftp_upload/4171/4171fig2.jpg" /> 图2。孤立的中脑多巴胺能神经纤维的夹层技术。解剖黑质（SN）的腹侧被盖区（VTA）的技术。 1。取出覆皮层和海马（这些结构已经在上面的图片中删除）。 2。做一个垂直切割分离色素面​​积的SN从腹侧被盖区。 3。使背外侧部分的SN以上达到或接近中线的横向切。 4。逗的SN离脑干休息。一旦SN已被删除，同样挑逗腹侧被盖区远离其余的脑。注：本节通常是在观察坐标，相对于前囟门在5.7美联社。 <img alt="图3" src="/files/ftp_upload/4171/4171fig3.jpg" /> 图3。第二 ​​（最后）的总蛋白负载丽春红S染色图像J分析正常化。丽春红S的大鼠黑质样品的总次测定的蛋白质染色。第一个五年车道形成的左侧是标准的总次曲线，以28μg蛋白大鼠肝匀浆被添加到镜像，从黑质样品的蛋白质负荷。旁边车道包含分子量（MW）的标记。大鼠黑质样品，其余车道包含〜28μg蛋白。根据每个样品的蛋白浓度BCA法测定蛋白质的负载。然而，尽管应该是平等的负载，有黑暗丽春红S染色之间的样本显示轻微的变化ImageJ的分析来分析每个样品丽春红S染色金额正常化，这是纠正的蛋白质含量的变异。 <img alt="图4" src="/files/ftp_upload/4171/4171fig4.jpg" /> 图4。代表TH控制和治疗的磷酸化的结果。代表ser31磷酸化酪氨酸羟化酶（PTH）和ser40生理盐水和甲基安非他明治疗大鼠PTH西方印迹，从先前的研究4。 SER 31 PTH在大鼠黑质样品。标定辑31 PTH标准曲线范围从0.3毫微克至2.0纳克，检测线性范围内。以前总TH水平的Western blot分析的基础上，每个样品装入6纳克总TH。 ser40 PTH在大鼠腹侧被盖样品标定ser40 PTH标准曲线的范围从0.2毫微克至1.5纳克，检测线性范围内。以前总TH水平的Western blot分析的基础上，9纳克总TH加载每个样品。

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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>

没有利益冲突的声明。

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 ...

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

Research

JoVE Journal

Neuroscience

25.6K 次观看.  Louisiana State University Health Sciences Center. 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。

视频: 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.

单细胞的多巴胺释放，同时电压钳和电流分析法测量

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

科研

神经科学

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.

在微RNA在腹鸡中脑大毛表达

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.

中脑多巴胺神经元的数目在成年小鼠环境的调变

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.

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

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.

高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价

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.

用光学分馏塔和标准显微镜设备对小鼠黑质多巴胺能神经元数的视学估计

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.

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

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.

凝胶神经元全细胞斑块夹的急性脊髓切片的制备

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

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.

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 ...