Chronic electrical stimulation induces functional network changes in cortical neuron cultures

Recent studies demonstrated that a functional brain network could be regarded as a complex network. With the help of network theory, neuroscientists can identify common organizational principles of the functional brain networks. As a consequence, some non-random organizational features, such as “small world” (most of the nodes are not connected directly but can communicate with few intermediate relay steps) and “rich club” (nodes that are rich in connections tend to form strongly interconnected clubs), have been found in functional brain network. Recently, the “small world” organizational feature of neuronal functional networks in vitro was found to be influenced by external applications. However, little is known about the influence of chronic electrical stimulation on functional networks of dissociated cortical cultures during network development. In the present study, cortical cultures were electrically stimulated at a frequency of 0, 0.02, and 0.2 Hz, between 7 and 26 days in vitro (DIV). The spontaneous activity of the cortical cultures was recorded using MEAs. Next, a cross-covariance method and graph theory were applied to investigate organizational feature of functional networks. Our results showed that over 3 weeks of stimulation, the network density significantly increased with maturation in the control and 0.02 Hz stimulation groups, but not in 0.2 Hz stimulation groups. Moreover, all the cultures had a small-world topology at 14, 18, 22, and 26 DIV, free from the effect of chronic electrical stimulation. Besides, we found an asymmetry effect that partial electrical stimulation inhibited the formation of node connections in stimulated areas. This effect was more pronounced at 0.2 Hz than at 0.02 Hz stimulation. Our results suggest that electrical stimulation does not affect the small-world properties of neural cultures. Instead, electrical stimulation modulates connectivity patterns, and neurons within the stimulated area are less connected than neurons outside the stimulated area.