Multi-electrode stimulation aims to induce sophisticated brain perception by eliciting specific spatial and temporal patterns of neural activity corresponding to sensory perceptions[1][2][3]. However, conventional implanted microelectrode stimulation often results in large-scale brain responses, from a few hundred microns to a few millimeters[2]. This spread of signals may lead to complex signal interactions and integration across adjacent electrodes.In our study, we positioned two monopolar micro-electrodes at various spacings ranging from 400 to 800µm between them, and simultaneously stimulated visual cortex of mice under anesthesia. We applied slightly above threshold level of amplitude ranging between 12~20uA with a stimulation frequency of 200Hz. Neuronal calcium responses were recorded using two-photon microscopy to examine single-cell responses and wide-field imaging for mesoscale responses.Comparing the response from two-electrode stimulation to single-electrode stimulation, we observed that a subset of neurons exhibited either significant increase or decreases in activity during two electrode stimulation. Additionally, we observed the activation of previously silent neurons during two-electrode stimulation. The number of neurons responding to two-electrode stimulation was substantially higher compared to single-electrode stimulation, with an increase of neuropil activity. Our results highlight the spatiotemporal dynamics of response modulation depending on the proximity of the stimulating sites and provide valuable insights into the optimal implementation of multi- electrode stimulation techniques.Chen et al., (2020) Science.Hao et al., (2016). Frontiers in neural circuits.Flesher et al., (2016). Science translational medicine.