Lau Thorsten, Proissl Verena, Ziegler Janina, Schloss Patrick
Biochemical Laboratory, Department of Psychiatry and Psychotherapy, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Germany.
Biochemical Laboratory, Department of Psychiatry and Psychotherapy, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Germany.
J Neurosci Methods. 2015 Feb 15;241:10-7. doi: 10.1016/j.jneumeth.2014.12.009. Epub 2014 Dec 18.
To study serotonergic volume neurotransmission at cellular level it needs to investigate neurotransmitter release and re-uptake sites in serotonergic neurons. However, due to the low number of cell bodies in the raphe nuclei and their widely branching neurites, serotonergic neuronal cultures are not accessible ex vivo.
We have combined differentiation protocols for the generation of stem cell-derived serotonergic neurons together with confocal microscopy to study the uptake and release of fluorescent substrates known to be selectively taken up by monoaminergic neurons. These substances include: (i) 4-(4-(dimethylamino)styryl)-N-methylpyridiunium (ASP+), an analog of the neurotoxin MPP+; (ii) the fluorescent false neurotransmitter (FFN511); and (iii) serotonin (5-hydroxytryptamine; 5-HT) itself, which is known to emit fluorescence upon excitation at 320-460nm.
ASP+ is taken up into living serotonergic neurons through the serotonin transporter, but not accumulated into synaptic vesicles; FFN511 diffuses in a SERT-independent way into serotonergic neurons and accumulated into synaptic vesicles. KCl-induced release of FFN511 and 5-HT can be visualized and quantified in living serotonergic neurons.
Application of ASP+ so far has been used to investigate substrate/transporter interactions; studies on FFN511 uptake and release have only been performed in dopaminergic neurons; quantitative studies on uptake and release of 5-HT in living serotonergic neurons have not been reported yet.
The differentiation protocols for the generation of stem cell-derived serotonergic neurons combined with the application of different fluorescent dyes allow to quantify neurotransmitter uptake and release in living serotonergic neurons in vitro.
为了在细胞水平研究5-羟色胺能容积性神经传递，需要研究5-羟色胺能神经元中的神经递质释放和再摄取位点。然而，由于中缝核中细胞体数量较少且其神经突广泛分支，无法通过离体方式获得5-羟色胺能神经元培养物。
我们将用于生成干细胞衍生的5-羟色胺能神经元的分化方案与共聚焦显微镜相结合，以研究已知被单胺能神经元选择性摄取的荧光底物的摄取和释放。这些物质包括：（i）4-（4-（二甲基氨基）苯乙烯基）-N-甲基吡啶鎓（ASP+），神经毒素MPP+的类似物；（ii）荧光假神经递质（FFN511）；以及（iii）5-羟色胺（5-羟色胺；5-HT）本身，已知其在320 - 460nm激发时会发出荧光。
ASP+通过5-羟色胺转运体被摄取到活的5-羟色胺能神经元中，但不会积累到突触小泡中；FFN511以不依赖于5-羟色胺转运体（SERT）的方式扩散到5-羟色胺能神经元中并积累到突触小泡中。KCl诱导的FFN511和5-HT的释放可以在活的5-羟色胺能神经元中可视化并定量。
到目前为止，ASP+的应用一直用于研究底物/转运体相互作用；关于FFN511摄取和释放的研究仅在多巴胺能神经元中进行；尚未有关于活的5-羟色胺能神经元中5-HT摄取和释放的定量研究报道。
用于生成干细胞衍生的5-羟色胺能神经元的分化方案与不同荧光染料的应用相结合，能够在体外定量活的5-羟色胺能神经元中的神经递质摄取和释放。
