Zimmer L, Kodas E, Guilloteau D, Garreau L, Besnard J, Chalon S
CERMEP Biomedical Cyclotron, Lyon, France.
J Neurosci Methods. 2000 Nov 30;103(2):137-44. doi: 10.1016/s0165-0270(00)00287-9.
The role of dopamine as a major modulator of CNS function is well-known, and the homeostasis of dopamine is considered to be of major importance in the pathogenesis of several psychiatric and neurological diseases. Few methods are currently available for in vivo study of dopamine transporter function, which regulates extracellular levels of dopamine. Adapting the 'indicator diffusion' method applied to the microdialysis technique, we present here a suitable method for this functional investigation. We measured the cellular extraction of [3H]-MPP+, which is known to accumulate in the dopaminergic neurones through the DAT in the rat striatum, using [14C]-mannitol as reference substance characterized by absence of cellular accumulation. The cellular extraction was 0.41 and was almost abolished in the presence of the dopamine-uptake inhibitor cocaine, reaching 0.07. This suggested that extraction of [3H]-MPP+ was due to cellular uptake by dopamine transporters. Tissue analysis confirmed that [3H]-MPP+ was internalized in cells and that such transport was stopped by cocaine. Moreover, [3H]-MPP+ extraction was dramatically decreased after lesioning the nigro-striatal pathway with 6-hydroxydopamine, whereas [14C]-mannitol extraction was unchanged. It is concluded that the presented method can be used to study the functioning of the dopamine transporter in live animals.
多巴胺作为中枢神经系统功能的主要调节因子，其作用已广为人知，多巴胺的稳态被认为在几种精神疾病和神经疾病的发病机制中至关重要。目前，用于多巴胺转运体功能体内研究的方法很少，而多巴胺转运体可调节细胞外多巴胺水平。通过将“指示剂扩散”方法应用于微透析技术，我们在此提出一种适用于该功能研究的方法。我们使用[14C]-甘露醇作为无细胞蓄积特征的参考物质，测量了[3H]-MPP+在大鼠纹状体中通过多巴胺转运体（DAT）在多巴胺能神经元中的细胞摄取。细胞摄取率为0.41，在多巴胺摄取抑制剂可卡因存在的情况下几乎被消除，降至0.07。这表明[3H]-MPP+的摄取是由于多巴胺转运体的细胞摄取。组织分析证实[3H]-MPP+被细胞内化，且这种转运被可卡因阻断。此外，用6-羟基多巴胺损伤黑质-纹状体通路后，[3H]-MPP+的摄取显著降低，而[14C]-甘露醇的摄取未改变。结论是，所提出的方法可用于研究活体动物中多巴胺转运体的功能。
