The functional transmission of information between different synapses is crucial, and the potential for such transmission in a mixed-cultured neural network of chick and rat has been demonstrated. We analyzed the correlation between neural electrical activity bursts using clustering and neural Ca2+ signal peaks obtained from Ca2+ imaging method. This analysis showed that information transmission occurs between chick neurons and rat neurons in a mixed-cultured neuronal network of chick and rat.We also cultured glial cells from both chickens and rats, combined with a glial cell-derived conditioned medium, and analyzed the effects of these glial cells derived from different species on neuronal networks. It has been suggested that glial cells derived from rats prolong the period of spontaneous neural electrical activity expression in cultured neural networks, and that direct cell contact is necessary. Moreover, the glial-derived conditioned medium influences the activity of spontaneous neural electrical activity, with rat glial-derived conditioned medium increasing the frequency of this activity in cultured neural networks.These results indicate that different types of neurons can form networks and transmit information, and that different types of glial cells can regulate the maintenance and activity of these neural networks. In particular, rat glial cells have been shown to activate the neural network by extending the maintenance period of the cultured chicken neural network and increasing the frequency of spontaneous neural electrical activity. The activation effect of the neural network by heterologous glial cells could potentially be useful in medical engineering.