Fang Huaifang, Pajski Megan L, Ross Ashley E, Venton B Jill
Dept. of Chemistry, University of Virginia, Charlottesville, VA 22904, (434) 243-2132.
Anal Methods. 2013;5(11):2704-2711. doi: 10.1039/c3ay40222c.
Methods to determine neurochemical concentrations in small samples of tissue are needed to map interactions among neurotransmitters. In particular, correlating physiological measurements of neurotransmitter release and the tissue content in a small region would be valuable. HPLC is the standard method for tissue content analysis but it requires microliter samples and the detector often varies by the class of compound being quantified; thus detecting molecules from different classes can be difficult. In this paper, we develop capillary electrophoresis with fast-scan cyclic voltammetry detection (CE-FSCV) for analysis of dopamine, serotonin, and adenosine content in tissue punches from rat brain slices. Using field-amplified sample stacking, the limit of detection was 5 nM for dopamine, 10 nM for serotonin, and 50 nM for adenosine. Neurotransmitters could be measured from a tissue punch as small as 7 µg (7 nL) of tissue, three orders of magnitude smaller than a typical HPLC sample. Tissue content analysis of punches in successive slices through the striatum revealed higher dopamine but lower adenosine content in the anterior striatum. Stimulated dopamine release was measured in a brain slice, then a tissue punch collected from the recording region. Dopamine content and release had a correlation coefficient of 0.71, which indicates much of the variance in stimulated release is due to variance in tissue content. CE-FSCV should facilitate measurements of tissue content in nanoliter samples, leading to a better understanding of how diseases or drugs affect dopamine, serotonin, and adenosine content.
为了绘制神经递质之间的相互作用图谱，需要有能够测定小组织样本中神经化学物质浓度的方法。特别是，将神经递质释放的生理测量值与小区域内的组织含量相关联将很有价值。高效液相色谱法（HPLC）是组织含量分析的标准方法，但它需要微升样本，而且检测器通常因被定量化合物的类别而异；因此，检测不同类别的分子可能会很困难。在本文中，我们开发了一种带有快速扫描循环伏安检测（CE-FSCV）的毛细管电泳法，用于分析大鼠脑片组织打孔样本中的多巴胺、5-羟色胺和腺苷含量。使用场放大进样堆积技术，多巴胺的检测限为5 nM，5-羟色胺为10 nM，腺苷为50 nM。神经递质可以从小至7μg（7 nL）组织的组织打孔样本中测量，这比典型的HPLC样本小三个数量级。对穿过纹状体的连续切片中的打孔样本进行组织含量分析，结果显示前纹状体中的多巴胺含量较高，但腺苷含量较低。在脑片中测量刺激后的多巴胺释放，然后从记录区域采集组织打孔样本。多巴胺含量与释放的相关系数为0.71，这表明刺激释放中的大部分差异是由于组织含量的差异所致。CE-FSCV应有助于测量纳升样本中的组织含量，从而更好地理解疾病或药物如何影响多巴胺､5-羟色胺和腺苷含量。
