体外观察直流电刺激（DCS）对神经元生长及突触连接超微结构的影响。

体外分离获得SD大鼠原代海马神经元并分为6组，分别施加0（对照组）、5、20、50、100及200 μA DCS进行干预，30 min/d，连续7 d。通过显微镜观察评估不同强度的恒定直流电干预第1、3、5、7天对神经元分化、突触生长长度及数量的影响；通过Western blot方法检测Synapsin和PSD95等突触蛋白表达；同时筛选体外DCS的最佳强度，并以此干预体外3D培养的神经元，通过神经突起生长染色检测海马神经元轴突生长活性；通过透射电镜技术评估在最适恒定DCS作用下体外培养的原代海马神经元的生长情况及对神经元之间形成突触连接结构的影响。

镜下明场观察发现，不同强度DCS下随着干预天数的增加，细胞轴突长度和数量均有明显增加，50 μA DCS促进原代海马神经元在体外分化效果最优，且神经元轴突数量较多、轴突生长能力较强。Western blot结果显示，5、20、50、100及200 μA DCS干预神经元的PSD95和Synapsin蛋白表达水平均高于对照组，差异有统计学意义（P<0.05）；其中50 μA DCS作用下原代海马神经元突触蛋白Synapsin和PSD95的表达水平显著高于其他组，提示50 μA为体外促进神经突触生长的最适电流强度。神经突起生长染色和透射电镜显示，50 μA DCS作用下3D培养的体外神经元的轴突生长活力强于对照组，且可以观察到更多的突触连接结构，包括类似增厚的膜结构和连接间隙。

50 μA DCS可促进体外3D培养的原代海马神经元突触样超微结构形成，其机制可能与上调了突触蛋白Synapsin和PSD95的表达水平有关。

To observe the effects of direct current stimulation (DCS) on neuronal growth and synaptic connectivity ultrastructure in vitro.

Primary hippocampal neurons from SD rats were obtained by isolation in vitro and divided into 6 groups. Each group was subjected to DCS intervention at 0 (control group), 5, 20, 50, 100, and 200 μA, respectively, for 30 min per day, continuously for 7 d. The effects of constant DCS at different intensities on neuronal differentiation, synaptic growth length and number were evaluated by microscopy on days 1, 3, 5, and 7. Synaptic protein expression including Synapsin and PSD95 was detected by Western blot. The optimal DCS intensity was selected and applied to neurons cultured in a 3D system. Axonal growth activity in hippocampal neurons was detected by neurite growth staining; and the growth of primary hippocampal neurons as well as the formation of synaptic connections under optimal DCS were observed using transmission electron microscopy.

Bright-field microscopy revealed that with increasing days of intervention, both the length and number of neuronal axons significantly increased under various DCS intensities. DCS at 50 μA better promoted the differentiation of primary hippocampal neurons in vitro, resulting in a higher number of axons and stronger axonal growth capacity. Western blot results showed that the expression levels of PSD95 and Synapsin proteins in neurons intervened with 5, 20, 50, 100, and 200 μA DCS were higher than those in the control group, and the differences were statistically significant (P<0.05); The expression levels of synaptic proteins Synapsin and PSD95 in primary hippocampal neurons under the action of 50 μA DCS were higher than those in other groups, indicating that 50 μA is the optimal current intensity for promoting synaptic growth in vitro. Neurite outgrowth staining and transmission electron microscopy demonstrated that under 50 μA DCS, neurons in 3D culture exhibited stronger axonal growth activity and more synaptic connection structures, including thickened membrane-like structures and synaptic clefts, compared to the control group.

Direct current stimulation at 50 μA promotes the formation of synapse-like ultrastructures in primary hippocampal neurons cultured in a 3D system in vitro, potentially through upregulating the expression of Synapsin and PSD95.