A novel dopamine transporter transgenic mouse line for identification and purification of midbrain dopaminergic neurons reveals midbrain heterogeneity.
A novel dopamine transporter transgenic mouse line for identification and purification of midbrain dopaminergic neurons reveals midbrain heterogeneity.
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DOI：
10.1111/ejn.13046
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发表时间：
2015-10
期刊：
影响因子：
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通讯作者：
Rickhag M
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文献类型：
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作者：
Apuschkin M;Stilling S;Rahbek-Clemmensen T;Sørensen G;Fortin G;Herborg Hansen F;Eriksen J;Trudeau LE;Egerod K;Gether U;Rickhag M
中脑多巴胺能（DA能）神经元是异质的，并且由投射到基底神经节、前额叶皮质和边缘系统的功能不同的细胞群组成。尽管DA能神经元具有重要的功能意义，但中脑DA能神经元的数量很少，在小鼠中由20- 30，000个神经元组成，因此需要开发新的工具来识别这些细胞。在这里，我们表征了细菌人工染色体（BAC）小鼠系（数据1-eGFP）基因表达神经系统图谱，表达增强的绿色荧光蛋白（eGFP）的多巴胺转运蛋白（DAT）启动子的控制下。脑切片的共聚焦显微镜分析显示在中脑区域和纹状体末端中与DA能标记物DAT和酪氨酸羟化酶（TH）共定位的强eGFP报告基因。在腹侧中脑中eGFP报告信号与DAT和TH的共定位的彻底定量表明，绝大多数eGFP表达神经元是DA能的。重要的是，表达谱也显示DA能异质性比较黑质（SN）和腹侧被盖区（VTA）。Dat 1-eGFP小鼠在纹状体和中脑中既没有显示突触体多巴胺摄取的变化，也没有显示DAT和TH水平的改变。我们发现Dat 1-eGFP和野生型之间没有行为差异，表明该菌株没有异常。最后，可以通过荧光激活细胞分选从出生后的小鼠获得高度富集DA能神经元的细胞群，并且可以在体外培养分选的神经元。我们的研究表明，在这个小鼠系中的eGFP表达是选择性的DA能神经元，这表明Dat 1-eGFP小鼠品系构成了一个有前途的工具，描绘多巴胺生物学的新方面。
Midbrain dopaminergic (DAergic) neurons are heterogeneous and composed of functionally distinct cell populations projecting to the basal ganglia, prefrontal cortex and limbic system. Despite their functional significance, the midbrain population of DAergic neurons is sparse, constituting 20–30,000 neurons in mice, and development of novel tools to identify these cells is warranted. Here, we characterize a bacterial artificial chromosome (BAC) mouse line (Dat1-eGFP) from Gene Expression Nervous System Atlas that expresses enhanced green fluorescent protein (eGFP) under control of the dopamine transporter (DAT) promoter. Confocal microscopy analysis of brain sections showed strong eGFP reporter in midbrain regions and striatal terminals that co-localized with the DAergic markers DAT and tyrosine hydroxylase (TH). Thorough quantification of co-localisation of the eGFP reporter signal with DAT and TH in the ventral midbrain showed that a vast majority of eGFP-expressing neurons are DAergic. Importantly, expression profiles also revealed DAergic heterogeneity when comparing substantia nigra (SN) and ventral tegmental area (VTA). Dat1-eGFP mice showed neither change in synaptosomal dopamine uptake nor altered levels of DAT and TH in both striatum and midbrain. We found no behavioral difference between Dat1-eGFP and wildtype suggesting that the strain is not aberrant. Finally, cell populations highly enriched in DAergic neurons can be obtained from postnatal mice by fluorescence-activated cell sorting and the sorted neurons can be cultured in vitro. Our investigation demonstrates that eGFP expression in this mouse line is selective for DAergic neurons, suggesting that the Dat1-eGFP mouse strain constitutes a promising tool for delineating new aspects of dopamine biology.