这项工作得到了 UCPH 2016 方案的支持 (U.G., ar, K.J.), Lundbeck 基金会 (大副) Lundbeck 生物膜基金会中心纳米 (U.G.), 国家卫生研究院赠款 P01 DA 12408 (U.G.), 丹麦独立研究委员会-医学科学 (U.G.)。

丹麦动物实验视察员的指导方针 (许可号: 2017-15-0201-01160) 被遵循, 并在当地动物福利监督下在一个完全 AAALAC 的认证设施中进行实验委员会. 
 1. 触多巴胺摄取量 (方法 1) 注意: 此协议用于两个大脑的并行评估, 但可以成功地用于执行触 DA 摄取实验与四大脑并行. 
<ol> <li> 准备 <ol> <li> 标签 48 1.5 毫升微型离心机管每 表 1 (每个大脑 24)。使用不同颜色的管属于每个大脑, 以防止混淆之间的样本 </li> <li> 标签51闪烁管 #1-51. </li>
		<li> 在冰上放置磷酸盐缓冲盐水 (PBS)。这将用于保持大脑的冷却. </li>
		<li> 打开离心机以允许 pre-cool 到4和 #176; C. </li>
		<li> 前称两个微离心管, 以获得确切的组织重量在触准备 (2.6 节). </li>
		<li> 通过混合4毫米 HEPES 和0.32 米蔗糖来制备均匀化缓冲。调整 pH 值为 7.4, 并保持冰. 
		注: 均匀化缓冲液可保存在等分-20 和 #176 ° C, 并解冻实验的当天. </li>
		<li> 准备吸收缓冲器的组合如下:25 毫米 HEPES, 120 毫米氯化钠, 5 毫米氯化钾, 1.2 毫米 CaCl 2 , 1.2 毫米 MgSO 4 , 1 毫米抗坏血酸 (0.194 克/升), 5 毫米 d-葡萄糖 (0.991 克/升)。用氢氧化钠调节 pH 值至 7.4 (导致钠浓度约130毫米), 并保持在冰上. 
		注: 对于2大脑, 准备1500毫升吸收缓冲。准备吸收缓冲液, 作为一个10x 的库存解决方案, 不含抗坏血酸和葡萄糖保持在4和 #176; c. 使1x 的工作解决方案新鲜的一天, 补充与抗坏血酸和葡萄糖。将 pH 值与氢氧化钠调整到 7.4. </li>
		<li> 准备吸收缓冲剂 + 配体:25 毫米 HEPES, 120 毫米氯化钠, 5 毫米氯化钾, 1.2 毫米 CaC l2 , 1.2 毫米 MgSO 4 , 1 毫米抗坏血酸 (0.194 克/升), 5 毫米 d-葡萄糖 (0.991 克/升), 1 和 #956; M pargyline, 100 nm 帕明, 10 nm邻苯二酚邻甲基转移酶 (COMT) 抑制剂。用氢氧化钠调节 pH 值 7.4, 保持冰层. 
		注: 使用50毫升吸收缓冲剂并加入配体。Pargyline 添加, 以防止氧化降解 DA 通过单胺氧化酶 (毛)。添加 COMT 抑制剂 (RO-41-0960), 以防止 DA (儿茶酚胺在一般) 的退化, 通过 COMT。帕明被添加到抑制通过去甲肾上腺素和血清素转运蛋白的摄取, 从而使摄取的结果特定的 DAT. </li>
		<li> 准备吸收缓冲剂 + 配体 + 可卡因的组合如下:25 毫米 HEPES, 120 毫米氯化钠, 5 毫米氯化钾, 1.2 毫米 CaCl 2 , 1.2 毫米 MgSO 4 , 1 毫米抗坏血酸 (0.194 克/升), 5 毫米 d-葡萄糖 (0.991 克/升), 1 和 #956; M pargyline, 100 nM 帕明, 10 nM COMT 抑制剂, 500 和 #956; 我的可卡因。用氢氧化钠调整 pH 值至 7.4, 并保持冰层. 
		注: 使用7毫升吸收缓冲 + 配体和添加可卡因。添加可卡因是为了获得对非特异 DA 摄取量的背景测量, 以便从数据中减去, 只留下特定于 DAT 的摄取量 (因为如上所述, SERT 和网络受到帕明的抑制)。另外, 一个高度有效的和特定的 DAT 抑制剂 GBR-12935, 可以改用. 
		要点: 从现在开始, 将所有的东西都放在冰上 . </li>
	</ol> </li> <li> 触准备 <ol> 一次牺牲一只老鼠由颈椎脱位和斩首. <li>
		<li> 用剪刀切开皮肤以露出颅骨, 并切断头骨后遗留下来的任何多余的组织。沿着 缝合放血 的小直剪刀将头骨切开, 尽可能前. </li>
		<li> 将两个剪刀尖放在鼠标的每只眼睛上, 然后将头骨切开, 以除去头骨和完整的大脑的剩余部分。迅速移除大脑并将其放置在 ice-cold PBS 中。这将使它保持低温, 而第二个大脑被解剖. </li>
		<li> 使用脑基质, 解剖纹状体的3毫米冠状切面 (前后: +1.5 毫米至-1.5 毫米), 然后用穿孔器进行更精细的解剖。请参见 图 1 以了解穿孔区域. </li>
		<li> 在任何时候保持组织在冰上前进. </li>
		<li> 将组织转移到1.5 毫升微离心管上进行称量. 
		注: 此重量将用于步骤 1.2.14. </li>
		<li> 对第二个大脑重复步骤 1.2. 1-1. 2.6. </li>
		<li> 一旦获得两个大脑的重量, 将组织转移到包含1毫升 ice-cold 均匀化缓冲液的匀质玻璃上. </li>
		<li> 质的组织使用马达驱动杵在 800 rpm, 10 甚至中风. 
		注意: 一个笔画等于一个上下动作, 第一个笔画应该是5秒, 下面3-4 秒. 等渗介质的均匀化对于防止突 6 的破裂非常重要。使用适当的力和等渗介质将允许前终端重新包围细胞质, 突触泡, 线粒体和细胞骨架 15 . </li>
		<li> 将匀浆转移到1.5 毫升微离心管上, 用0.5 毫升的均匀化缓冲液冲洗均匀玻璃中的匀浆. </li>
		<li> 保持样品在冰上, 而来自其他大脑的组织均匀。在步骤 1.2. 12-1. 2.13 中并行运行两个大脑. </li>
		<li> 用离心法 (1000 x g, 10 分钟, 4 和 #176; C). </li>
		<li> 将上清液 (S1) (含细胞膜和细胞质) 转移到新的1.5 毫升离心管和离心机在 1.6万 x g 为20分钟在4和 #176; C. </li>
		<li> 在40和 #956 中丢弃上清 (含细胞质) 和重颗粒 (P2) (含粗突); L 均质缓冲/镁组织 (基于步骤1.2.6 中获得的重量)。用 p1000 吸管轻轻重粗触馏分, 因为太多的力会破坏突. 
		注意: 重要的是要结束至少280和 #956;如果没有, 稀释超过40和 #956; L 每毫克将是必要的。步骤 1.2. 1-1. 2.13 必须尽可能快地进行, 所有的东西都要放在冰上。一旦原油突已悬浮在均匀化缓冲区, 他们是相当稳定的, 只要他们保持在冰上 6 , 15 , 16 . </li>
	</ol> </li> </ol> 2。摄取实验 <ol> <li> 添加440和 #956; 在指定的 12 1.5 毫升离心管和440和 #956 中吸收缓冲 + 配体 + 可卡因; l 吸收缓冲 + 配体到36剩余的 1.5 ml 离心管 (参见 表 1 进行布局). 
	注意: 在实验开始之前, 从冰中取出15分钟把他们带到室温, 但保持冰, 直到然后保存抑制剂. </li>
	<li> 准备饱和度曲线 (多巴胺 (2, 5, 6-[3 h]-DA) (3-h 多巴胺) (91.1 Ci/摩尔) 在不同的最终浓度 (0.031, 0.0625, 0.125, 0.25, 0.5, 和1.0 和 #956; M). 
	<ol> <li> 标签六 2 mL 离心管作为10、5、2.5、1.25、0.62 和 0.31. </li>
		<li> 添加750和 #956; L 吸收缓冲 + 配体到5离心管, 最低数量. </li>
		<li> 添加1455.4 和 #956; l 吸收缓冲 + 配体, 14.6 和 #956; l 多巴胺 (1 毫米), 30 和 #956; l 3-H 多巴胺 (11 和 #956; M) 到被标记的管子 10. </li>
		<li> 传输750和 #956; L 从被标记的管子10到被标记的管子5。混合好, 并转移750和 #956; L 从这个管到2.5 管。重复这种稀释与其余的管. </li>
	</ol> </li> <li> 在将玻璃纤维过滤器放在12井过滤器桶上后, 用4毫升的吸收缓冲液进行预冲洗. </li>
	<li> 添加10和 #956; 触膜悬浮液对前 24 1.5 毫升微离心管 (见 表 1 用于布局) 包含440和 #956; l 缓冲器。涡旋小心, 并在短期内降速, 以确保突被淹没在缓冲区。在 37 o C 处保留10分钟 
	注意: 重要的是要准确的时间在吸收实验期间, 公平比较大脑。使用秒表来精确. </li>
	<li> 添加50和 #956; 浓度为10和5的多巴胺和 #956; M (2.2 节) 到前两列, 并在37点处离开, C 为5分钟, 并晃动。在整整5分钟后, 通过增加1毫升 ice-cold 吸收缓冲来停止反应。对浓度2.5 和1.25 和 #956 进行同样的处理; m (第 2.2), 然后集中0.62 和0.31 和 #956; m. </li>
	<li> 将样品添加到 pre-rinsed 超细纤维过滤器中, 并用 5 x 4 mL ice-cold 吸收缓冲器清洗。当前12样本被添加到滤镜中时, 将滤镜移至闪烁管。在接下来的12示例中重复此操作. </li>
	<li> 对下一个大脑重复步骤 2.4-2.6. </li>
	<li> 在闪烁管的底部放置一个超细纤维过滤器, 并在顶部添加25和 #181, 从而准备3最大计数管; 最大多巴胺浓度 (10 和 #956; M). </li>
	<li> 将所有51闪烁管放在通风罩中1小时. </li>
	<li> 在每个闪烁管上添加3毫升闪烁液, 并在1h 振动器上晃动. </li>
	<li> 计数 [ 3 H]-在 beta 计数器中为 1 min. 
	<ol> <li> 打开程序并选择: 标签: H-3, 板/过滤: 4 毫升瓶, 4 6, 化验类型: 正常和计数时间: 1 分钟 
		注意: 此步骤可在次日执行, 如果更方便的话。把样品用锡纸盖住过夜. </li>
	</ol> </li> <li> 蛋白质测定 <ol> <li> 使用标准的性能分析蛋白测定试剂盒来确定突的蛋白质浓度, 以便从每分钟数 (cpm) 到 fmol/分钟/#956 的调整; g 和适当的摄取比较在示例之间. </li>
	</ol> </li> </ol> 3。数据分析 <ol> <li> 使用吸收实验中的数据对每个组进行饱和度曲线, 并计算反应速率 (V max ) 和蔑氏常量 (K M ). 
	注意: K M 值反映达到 V max 的一半所需的基底浓度。因此, V max 直接反映了 dat (最大吸收容量) 的函数, 它取决于表面上的 dat 数以及它的活动如何通过后的修改和/或相互作用的蛋白质进行调制, 以及离子梯度的变化。K M 值是对传送器的基底亲和性的间接测量, 它也可以通过后的修改和/或相互作用的蛋白质进行调制。因此, 要完全评估功能变化, 就必须直接确定表面表达水平, 例如表面法测定。对多巴胺再的描述以及对 K M 和 V max 值的含义的阐述已经被 al. 17 . </li>
</ol> 4。高效液相色谱法 (方法 2) <ol> <li> 脑解剖 <ol> <li> 标签和称离心管; 每个区域每只鼠标一个. </li>
		<li> 一次性牺牲一只老鼠, 由颈部脱位和斩首。如1.2 节所述, 迅速移除大脑。立即进行进一步的解剖. </li>
		<li> 使用脑基质, 解剖一个3毫米的纹状体的冠状切片, 其次是 NAc 和 dStr 的更精细的双边解剖。请参见 图 3 以了解穿孔区域. </li>
		<li> 将组织转移到1.5 毫升离心管, 并迅速放置在干冰上。称量组织并立即将其放回干冰中. 
		注: 组织重量对以后的浓度计算很重要. </li>
		<li> 使用下一鼠标重复步骤 4.1. 1-4. 1.4. 
		注意: 将组织放置在 80 o C 上, 直到进一步处理或立即进行组织处理. </li>
	</ol> </li> </ol> 5。组织准备 <ol> <li> 打开离心机以使其 pre-cool 到4和 #176; C </li> <li> 准备均匀化解决方案 (0.1 N 高氯酸 (HClO 4 )) 并保持在冰上. </li>
	<li> 使用均匀化解决方案, 准备一个新的 0.1 pmol/和 #956; L 多巴胺标准从1毫米的股票溶液 (1万 x 稀释). 
	注: 库存溶液的制备方法是将多巴胺溶解于均匀缓冲液中, 以1毫米的浓度储存, 可保存在20和 #176; C 约一个月。如果大脑的其他区域感兴趣, 标准的集中将必须被修改, 因为其他大脑区域包括 prefrontral 皮层、海马、黑质和腹盖区域比纹状体拥有多达100倍的少 DA. </li>
	<li> 在每个样本中添加500和 #956、L 均质化解决方案 (, 即 NAc 和 dStr 组织; 第4.1 节) 和质样品, 使用 ultra-homogenizer 全速在大约三十年代. 在均匀化过程中保持样品在冰水中。在每个样品之间, 用清水清洗超匀浆机 (三十年代的全速). </li>
	<li> 离心样品30分钟在4和 #176; C, 1.4万 x g. </li>
	<li> 传输大约200和 #956; 在玻璃0.22 和 #956 上取样; m 滤镜 (1 厘米直径), 使用1毫升塑料注射器. 
	注: 玻璃过滤器的使用不重要, 只要选择的过滤器是0.22 和 #956; m 除去高分子量物质. </li>
	<li> 用电化学检测 (EC)-HPLC 法分析样品 18 如第6节所述. </li>
</ol> 6。高效液相色谱分析 <ol> <li> 为移动阶段准备以下解决方案: 醋酸钠55毫米, 磺酸1毫米, Na 2 EDTA 0.1 毫米, 乙腈 8%, 乙酸 0.1 M, pH = 3.2. 
	注: 用0.1 米醋酸调节 pH 值。准确地使用 pH 值是很重要的。解决方案应 degassed 由 on-line 气 (HPLC 系统的综合部分)。使 2 L 的移动阶段, 并改变它大约每月一次 (改变它时, 噪音得到明显)。由于波动, 它需要约24小时调整后, 改变移动阶段. </li>
	<li> 安装检测器: <ol> <li> 将电压输出设置为700毫伏, 温度为32和 #176; C 在探测器烤箱上; 这是非常重要的。使用在线手册制作一个范围的程序。请参阅手册以了解详细信息)。按 表 2 添加时间程序. 
		注: 在本研究中, 该程序设置为自动改变电流在设定的保留时间, 以保持 HPLC 在最佳电流和保持色谱的峰值尽可能扩大, 但仍在视野内。这是优化了纹脑裂解从小鼠. </li>
	</ol> </li> <li> 当程序已添加到检测器中时, 通过注入标准三次 (5、10和15和 #956; L). 
	注: 标准 (10 和 #956; l (10 和 #956; l x 0.1 pmol/和 #956; l = 1 pmol DA)) 应每第十样品注射一次, 并将样品校准到最接近的标准。微调系统的前一天, 调整3点标准, 并检查是否色谱在预期范围内出来。如果观察到大量噪音, 则更改移动阶段。如果一个峰值大于 990 mV, 然后重新在一个较低的体积样本, 或做一个新的范围程序和新的标准曲线。检测限制被测量为3倍的噪声值的 mV, 以最低峰值为每个标准注入的值 (NA, DOPAC, DA, 5-5-hiaa, HVA, 3 MT 和 5 HT)。
	通过打开 LC 解决方案并激活分析, <ol> <li> 设置系统 1; 这从联机模式开始。键入用户 ID 和密码, 然后单击 "确定"。等待 LC 解决方案连接到仪器。转到批处理表并填写数据信息 ( 例如 , 示例类型: 未知的样本和标准的 std, 分析类型: 它 QT, 注射量:10, ISTD amt: 1 级骗局)。保存文件 (转到文件和 #62; 将批处理文件保存为 #62; 保存). </li>
		<li> 通过按下和 #39 使系统注入第一个样本; 启动批处理和 #39;。这将导致注入10和 #956; L 样品由样与溶剂交付模块. 
		注: 使用0.15 毫升/分钟的泵流量和反转相的 C18 柱。这里使用了电化学检测的安培检测器。玻碳电极应设置为 0.8 V 与银/氯化参考电极。为了测定多巴胺, 使用色谱3和 #181; ODS (3) C18 (DA 2 mm x 100 mm, 粒度3和 #181; m) 来实现色谱分离. </li>
		<li> 提取记录和计算的峰值区域。
		当样本完成时, <ol> <li> 转到数据获取以跟踪色谱。转到 Lc 后运行分析-和 #62; 选择文件名 (色谱将打开)-和 #62; 单击 "查看"。在手动集成栏上, 添加所有要集成的峰值 #62; 转到数据报表并打印读出. 
			注: LC 解决方案软件用于数据分析和系统控制. </li>
		</ol> </li> </ol> </li> <li> 从峰值区域计算样本中多巴胺的浓度, 如下所示, 使用标准的已知浓度: <ol> <li> 使用标准的峰值区域 (等于 1 pmol) 来获取因子 a. 
		<img alt="Equation" src="/files/ftp_upload/56093/56093eq1.jpg"/> </li> <li> 使用因子 A 获取样本的浓度. 
		样品浓度 (pmol 每10和 #956; L) = 因子 A 和 #215; 示例的峰值区域 </li> <li> 使用此公式获取 ng 中的样本浓度. 
		样品浓度 pmol/10 和 #181; L x 500 和 #181; L x 兆瓦多巴胺 = 样品集中在 ng 样品浓度 (ng) = 样品浓度 (在 pmol 每10和 #956; l) 和 #160; x 500 和 #956; x 兆瓦多巴胺 </li> <li> 使用 ng 中的样品浓度得到 t他的样本浓度在 pg/g 组织 (样本浓度在 pg/组织 g = pg/g 组织). </li>
	</ol>
	</li>
</ol>

<ol>
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作者没有什么可透露的。

这篇手稿描述了有用的实验协议, 以描绘 DA 稳态在任何鼠标模型的选择。我们提供了详细的协议, 以测量 da 的脑组织从小鼠使用 HPLC 和触 da 吸收评估功能 da 运输通过 DAT。下面将阐述 HPLC 实验和触 DA 吸收法的程序、协议和限制。
触吸收协议可以为 dat 的功能提供有用的洞察力. 结合表面法实验13, 可以获得关于 dat 的总数量、表面级别和功能的知识。考虑到 da...

多巴胺 (DA) 是一种调节神经递质关键的运动行为, 奖励和认知功能1,7,8,9。DA 稳态失衡与一些神经精神疾病有牵连, 如注意缺陷多动障碍、药物成瘾、抑郁症和帕金森氏病1。DA 是释放从前神经元进入突触裂, 它绑定到和激活受体的前突触后膜, 从而进一步传递信号。在释放后突触中 DA 的水平由 DAT3,10进行空间和世俗控制。运输者固 da 从细胞外空间, 从而维持生理 da 水平3,11。在小鼠中, hyperdopaminergic 的基因去除导致突触 da 水平升高, 细胞内 da 池衰竭, 突触后 DAergic 信号发生深刻变化,10,12。
这里, 提出了两个单独的协议, 一个测量 DA 组织含量的方法, 另一个是评估 DAT 的功能. 结合加百利 et al. 的表面法测定法13这两种方法提供了有关 da内容和功能级别的 DAT, 以彻底评估 DA 稳态。利用这些方法可以对各种转基因小鼠或疾病模型的 DA 稳态进行定性和描述。这些工具已被实施和优化, 并在我们的实验室标准使用。目前的化验结果, 以调查的影响 da 稳态改变的 C 终端的 DAT14或表达的重组在酪氨酸羟化酶 (TH) 启动子5。

<table>
	<tbody>
		<tr>
			<td>COMT inhibitor</td>
			<td>Sigma Aldrich, Germany</td>
			<td>RO-41-0960</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>[3H]-Dopamine</td>
			<td>Perkin-Elmer Life Sciences, Boston, MA, USA</td>
			<td>NET67-3001MC</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Glass microfiber filters</td>
			<td>GF/C Whatman, GE Healthcare Life Sciences, Buckinghamshire</td>
			<td>1822-024</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>HiSafe Scintillation fluid</td>
			<td>Perkin Elmer</td>
			<td>1200-437</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>MicroBeta2</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>BCA Protein Assay kit</td>
			<td>Thermo Scientific Pierce</td>
			<td>23225</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Life Science</td>
			<td>H3375</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Life Science</td>
			<td>S7903</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Life Science</td>
			<td>S3014</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Life Science</td>
			<td>P9541</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Merck KGaA</td>
			<td>10043-52-4</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma Life Science</td>
			<td>63065</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma Life Science</td>
			<td>A0278</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma Life Science</td>
			<td>G7021</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Pargyline</td>
			<td>Sigma Aldrich</td>
			<td>P-8013</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Desipramine</td>
			<td>Sigma Aldrich</td>
			<td>D3900</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Dopamine</td>
			<td>Sigma Life Science</td>
			<td>H8502</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Cocaine</td>
			<td>Sigma Life Science</td>
			<td>C5776</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Brain matrix</td>
			<td>ASI instruments</td>
			<td>RBM2000C</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Cafano mechanical teflon disrupter</td>
			<td>Buch &#38; Holm</td>
			<td>Discontinued</td>
			<td>For synaptosomal DA uptake protocol (homogenization)</td>
		</tr>
		<tr>
			<td>Antec Decade (Amperometric detector)</td>
			<td>Antec, Leiden, The Netherlands</td>
			<td>Discontinued: new model DECADE Elite / Lite&#8482; Electrochemical Detector type 175 and 176</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Avantec 0.22 &#956;m glass filter</td>
			<td>Frisenette ApS, Denmark</td>
			<td>13CP020AS</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Column: Prodigy 3 &#956; ODS-3 C18</td>
			<td>Phenomenex, YMC Europe, Chermbeck, Germany</td>
			<td>Part Number:00A-3300-E0</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>LC solution software</td>
			<td>Shimadzu</td>
			<td>LabSolutions Series Workstation</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Perchlor acid 0.1M</td>
			<td>Fluka Analytical</td>
			<td>35418-500ml</td>
			<td>For HPLC protocol (Tissue preparation)</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E5134-50g</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Natriumdihydrogenphosphar</td>
			<td>Bie&#38;Berntsen</td>
			<td>1.06346 1000g</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Sodium 1-octanesulfonate monohydrate</td>
			<td>Aldrich</td>
			<td>74885 -10g</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Acetonitrile, isocratic HPLC grade</td>
			<td>Scharlau</td>
			<td>AC03402500</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Filtre 0.22um</td>
			<td>Frisenette ApS, Denmark</td>
			<td>Avantec 13CP020AS</td>
			<td>For HPLC protocol (Tissue preparation)</td>
		</tr>
		<tr>
			<td>ortho-Phosphoric acid 85%</td>
			<td>Merck</td>
			<td>1.00563. 1000ml</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Electrode</td>
			<td>Antec, Leiden, The Netherlands</td>
			<td>AN1161300</td>
			<td>For HPLC protocol (see manual online)</td>
		</tr>
		<tr>
			<td>Detector program on DECADE II electrochemical detector</td>
			<td>Antec, Leiden, The Netherlands</td>
			<td>Lite&#8482; Electrochemical Detector type 175 and 176</td>
			<td>For HPLC protocol</td>
		</tr>
	</tbody>
</table>

assessment of dopaminergic homeostasis in mice by use of high-performance liquid chromatography analysis and synaptosomal dopamine uptake

多巴胺 (DA) 是一种调节神经递质控制运动活动, 奖励过程和认知功能。多巴胺能 (DAergic) 神经与一些中枢神经系统相关疾病如帕金森氏症、注意力缺陷多动障碍和药物成瘾有密切关系1,2 ,3,4。划定疾病机制涉及 da 失衡是严重依赖于动物模型, 以模拟疾病的方面, 因此, 评估 da 稳态的特定部分的协议是重要的提供新的见解和可能的治疗这些疾病的目标。
在这里, 我们提出两个有用的实验协议, 当结合提供一个功能出的 DAergic 系统的小鼠。通过 da 水平和多巴胺转运器 (DAT) 功能的评估, 获得了 da 稳态的生物化学和功能参数5。在调查 da 系统时, 从成人大脑中可靠地测量 da 的内在水平的能力是必不可少的。因此, 我们提出如何对小鼠脑组织进行高效液相色谱 (HPLC) 测定 DA 的含量。我们执行的实验, 从背纹状体 (dStr) 和伏隔核 (NAc), 但该方法也适用于其他 DA 支配脑区。
DAT 是再进入前终端的关键, 从而控制了 da 的时间和空间活动。在评估 da 稳态时, 了解纹状体中的 DAT 的水平和功能是很重要的。在这里, 我们提供了一个协议, 允许使用触的6 DA 吸收法同时推断表面层和功能的信息。
目前的方法结合标准的免疫协议, 为研究人员提供了相关的 DAergic 系统的描述工具。

当前 DA 摄取协议 (图 1) 包括评估突中的 DAT 功能所需的所有步骤。我们的 DA 吸收方法 (图 2) 的代表性数据描述了带有未调整数据的饱和曲线 (图 2B) 和调节的数据 (图 2A)。饱和曲线显示野生型小鼠的摄取量。通常一个会使 DA 吸收与突变的小鼠相比, 这将导致一个饱和曲线为每个基因?...

Watch this Scientific Journal Video about Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake at JoVE.com

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

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

触多巴胺摄取和高效液相色谱分析是通过评估多巴胺转运体的功能和纹组织中多巴胺的水平来研究小鼠体内多巴胺稳态的实验工具,分别.在这里, 我们提出了测量多巴胺组织含量和评估多巴胺转运体功能的协议。

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

assessment-dopaminergic-homeostasis-mice-use-high-performance-liquid

Research

JoVE Journal

Neuroscience

12.3K 次观看.  University of Copenhagen. 触多巴胺摄取和高效液相色谱分析是通过评估多巴胺转运体的功能和纹组织中多巴胺的水平来研究小鼠体内多巴胺稳态的实验工具,分别.在这里, 我们提出了测量多巴胺组织含量和评估多巴胺转运体功能的协议。

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

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the beam.

在小鼠体内的汽车平衡和协调的评估使用的平衡木

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

科研

神经科学

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

T-迷宫强迫交替和评估工作，并参考在小鼠记忆的左，右的歧视任务

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

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.

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 ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

评估神经退行性表型<em&gt;果蝇</em&gt;爬坡试验和全脑多巴胺能神经元免疫组化染色

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. 
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.

We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of na&#239;ve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.

吗啡诱发的痛觉过敏和镇痛耐受小鼠中利用热和机械伤害性的评估方式

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular ...

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

高含量的成像检测方法的肉毒毒素抑制剂的鉴定

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

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, ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

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.

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

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