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>
	<li>Tritsch, N. X., Sabatini, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+modulation+of+synaptic+transmission+in+cortex+and+striatum.">Dopaminergic modulation of synaptic transmission in cortex and striatum.</a> Neuron. 76, 33-50 (2012).</li><li>Cartier, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biochemical+and+functional+protein+complex+involving+dopamine+synthesis+and+transport+into+synaptic+vesicles.">A biochemical and functional protein complex involving dopamine synthesis and transport into synaptic vesicles.</a> J Biol Chem. 285, 1957-1966 (2010).</li><li>Kristensen, A. 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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 &amp;amp; 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&amp;trade; Electrochemical Detector type 175 and 176</td><td>For HPLC protocol</td></tr><tr><td>Avantec 0.22 &amp;mu;m glass filter</td><td>Frisenette ApS, Denmark</td><td>13CP020AS</td><td>For HPLC protocol</td></tr><tr><td>Column: Prodigy 3 &amp;mu; 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&amp;amp;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&amp;trade; 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

Universidad de Cartagena UDEC

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 Sensorimotor Function in Mouse Models of Parkinson's Disease

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

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

帕金森氏症小鼠模型的感觉功能评估

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

科研

行为学

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

1单侧损毁6  - 羟基多巴胺帕金森病小鼠模型的发展

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

医学

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

突触前多巴胺纹状体脑片的快速扫描循环伏安动态

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

神经科学

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

用高压液相色谱法提取和分析儿茶酚胺神经递质及其代谢物的简便方法

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

化学

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 ...

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 ...

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

雌性叙利亚仓鼠自然奖赏行为中细胞外神经递质的测量体内变化

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

使用自动图像分析对啮齿动物模型的亚斯坦蒂娅尼格拉多巴明神经元密度进行半定量测定

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

一种基于板的测定法，用于测量急性脑切片中内源性单胺释放

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...

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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