Supplementary Material for: 5-Hydroxytryptamine 1A and 2B Serotonin Receptors in Neurite Outgrowth: Involvement of Early Growth Response Protein 1

Neurotransmitters play important roles in neurogenesis; in particular, acetylcholine and serotonin may regulate neurite elongation. Acetylcholine may also activate transcription factors such as early growth response protein 1 (EGR-1), which plays a role in neurite extension. N18TG2 neuroblastoma cells (which do not produce neurotransmitters and constitutively express muscarinic acetylcholine receptors) were transfected with constructs containing the cDNA for choline acetyltransferase, 5-hydroxytryptamine 1A (5-HT1A) and 5-HT2B serotonin receptors to study acetylcholine and serotonin interplay in neurite outgrowth. 5-HT1A receptor stimulation causes a decrease in EGR-1 levels and inhibition of neurite outgrowth; 5-HT2B stimulation, however, has no effect. Muscarinic cholinergic stimulation, on the other end, increases EGR-1 levels and fiber outgrowth. Inhibition of EGR-1 binding reduces fiber outgrowth activity. When both cholinergic and 5-HT1A receptors are stimulated, fiber outgrowth is restored; therefore, acetylcholine counterbalances the inhibitory effect of serotonin on neurite outgrowth. These results suggest that EGR-1 plays a role in the interplay of acetylcholine and serotonin in the regulation of neurite extension during development.神经递质在神经发生过程中发挥关键调控作用；其中，乙酰胆碱（acetylcholine）与血清素（serotonin）可参与调控神经突起伸长。乙酰胆碱还可激活早期生长应答蛋白1（early growth response protein 1, EGR-1）等转录因子，该蛋白在神经突起延伸过程中具有重要功能。 本研究选用无法合成神经递质且组成型表达毒蕈碱型乙酰胆碱受体的N18TG2神经母细胞瘤细胞，通过转染携带胆碱乙酰转移酶（choline acetyltransferase）、5-羟色胺1A型（5-hydroxytryptamine 1A, 5-HT1A）及5-HT2B型血清素受体的互补DNA（complementary DNA, cDNA）的重组构建体，以探究乙酰胆碱与血清素在神经突起生长过程中的相互作用。 实验结果显示，5-HT1A受体刺激会降低EGR-1的表达水平并抑制神经突起生长；而5-HT2B受体刺激则无显著调控效应。与之相反，毒蕈碱型胆碱能刺激可提升EGR-1的表达水平并促进神经纤维生长。抑制EGR-1的结合活性会减弱神经纤维的生长能力。当同时激活胆碱能与5-HT1A受体时，神经纤维生长可得到恢复，这表明乙酰胆碱能够抵消血清素对神经突起生长的抑制作用。 综上，本研究结果提示EGR-1参与了发育阶段中乙酰胆碱与血清素相互作用调控神经突起延伸的过程。