In vitro neuronal cultures have become a popular method with which to probe network-level neuronal dynamics and phenomena in controlled laboratory settings. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Here we demonstrate the effects of a high frequency electrical stimulation signal in training cultured networks of cortical neurons. Networks receiving this training signal displayed a time-dependent increase in the response to a low frequency probing stimulation, particularly in the time window of 20–50 ms after stimulation. This increase was found to be statistically significant as compared to control networks that did not receive training. The timing of this increase suggests potentiation of synaptic mechanisms. To further investigate this possibility, we leveraged the powerful Cox statistical connectivity method as previously investigated by our group. This method was used to identify and track changes in network connectivity strength.