Communication between astrocytes and neurons plays a pivotal role in the development, maintenance and dysfunction of cellular networks within the central nervous system. Further understanding the contribution of astrocytes to the initiation and progression of neuropathology has, however, been hindered by the complexity of cell networks in traditional in vivo and ex vivo systems. We have designed and constructed a novel three-compartment microfluidic cell culture device that enables us to overcome these limitations and uncouple the roles of astrocytes and neurons in the transfer of pathology through complex cell networks. Our microfluidic device integrates a novel maze-like structure that prevents synaptic connectivity between two fluidically isolated neuron populations, while allowing astrocyte infiltration and growth throughout. We use primary neuron/astrocyte co-cultures, proteomic analysis and immunocytochemistry to validate the application of this device. Using this device, we identify and describe a novel calcium-dependent role for astrocytes in the transfer of excitotoxic pathology between the segregated neuron populations.