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.