THE INVESTIGATION OF THE THERAPEUTIC MECHANISMS OF ANTIDEPRESSANT ON NEUROTRANSMITTER RELEASE

我们分子克隆了707个cDNA片段作为表达序列标签（est），并将其命名为抗抑郁相关基因adrg# 1-707。利用这些cDNA，我们开发了我们的原始cDNA微阵列，用于快速二次筛选候选基因作为新的治疗靶点。通过这个微阵列，我们发现在长期使用选择性血清素再摄取抑制剂抗抑郁药sertraline治疗的大鼠额叶皮层中，adrg# 14基因的表达显著增加。** adrg# 14与大鼠synaptobrevin 2 （vamp2）基因具有显著的同源性。为了进一步研究内源性VAMP2在神经生长因子（NGF）分化的PC12细胞中的功能作用，我们将VAMP2抗体转染到这些细胞中以抑制内源性VAMP2。用高[K+]缓冲液刺激细胞引起对照组[3H]去甲肾上腺素释放的时间依赖性升高。然而，这种释放在转染中显著而强烈地减少。另一方面，形态学变化的测量表明，每个细胞中最长的神经突的平均长度和每个细胞中神经突的总长度在转染后分别显著缩短。此外，每个细胞的平均神经突数在转染后也显著减少。这些数据表明，内源性VAMP2不仅在神经递质释放中起重要作用，而且在ngf分化的PC12细胞中对神经突的生长和发芽也起重要作用。先前，我们已经确定VAMP2作为抗抑郁药的新分子靶点。抗抑郁药的长期治疗作用之一可能与功能和结构神经可塑性的改变有关。

We have molecularly cloned 707 cDNA fragments as expressed sequence tags (ESTs), which we named them antidepressant related genes, ADRG#1-707. Using these cDNAs, we developed our original cDNA microarray for rapid secondary screening of candidate genes as the novel therapeutic targets. With this microarray, we found that the expression of the gene, ADRG#14 was significantly increased in rat frontal cortex which had b*** chronically treated with a selective serotonin reuptake inhibitor antidepressant, sertraline. Sequence analysis o**ADRG#14 with the EMBL/GeneBank database showed significant homology to rat synaptobrevin 2 (VAMP 2) gene. To investigate further the functional roles of endogenous VAMP2 in nerve growth factor (NGF)-differentiated PC12 cells, VAMP2 antibody was transfected to these cels to inhibit the endogeneous VAMP2. Stimulation of the cells with high-[K+] buffer caused a time-dependent elevation of [3H]noradrenaline release in controls. However, this release was significantly and strongly diminished in transfectants. On the other hand, measurement of the morphological changes demonstrated that the mean length of the longest neurite in each cell and the total length of neuiites per cell was significantly shorter in the transfectants, respectively. In addition, the mean neurite number per cell was also significantly smaller in the transfectants. These data indicated that endogenous VAMP2 plays an importanat role not only for neurotransmitter release, but also for neurite outgrowth and sprouting in NGF-differentiated PC12 cells. Previously, we have identified VAMP2 as a novel molecular target for antidepressant. One of the long-term therapeutic action of antidepressant may be related to the changes of both functional and structural neuroplasticity.