Author Summary Astrocytes communicate bidirectionally with neurons, enhancing synaptic plasticity and promoting the synchronization of neuronal microcircuits. We constructed a multilayer network model of neuron-astrocyte connectivity based on calcium activity in mixed cortical cultures, and used this model to evaluate the effect of glutamatergic inhibition and mechanical injury on network topology. We found that injury increased the strength, clustering, and efficiency of neuronal but not astrocytic networks, an effect that was not observed in injured networks pretreated with a glutamate receptor antagonist. Our findings demonstrate the utility of multilayer network models for characterizing the multiscale connectivity of two distinct but functionally dependent cell populations. Abstract Astrocytes communicate bidirectionally with neurons, enhancing synaptic plasticity and promoting the synchronization of neuronal microcircuits. Despite recent advances in understanding neuron-astrocyte signaling, little is known about astrocytic modulation of neuronal activity at the population level, particularly in disease or following injury. We used high-speed calcium imaging of mixed cortical cultures in vitro to determine how population activity changes after disruption of glutamatergic signaling and mechanical injury. We constructed a multilayer network model of neuron-astrocyte connectivity, which captured distinct topology and response behavior from single-cell-type networks. mGluR 5 inhibition decreased neuronal activity, but did not on its own disrupt functional connectivity or network topology. In contrast, injury increased the strength, clustering, and efficiency of neuronal but not astrocytic networks, an effect that was not observed in networks pretreated with mGluR 5 inhibition. Comparison of spatial and functional connectivity revealed that functional connectivity is largely independent of spatial proximity at the microscale, but mechanical injury increased the spatial-functional correlation. Finally, we found that astrocyte segments of the same cell often belong to separate functional communities based on neuronal connectivity, suggesting that astrocyte segments function as independent entities. Our findings demonstrate the utility of multilayer network models for characterizing the multiscale connectivity of two distinct but functionally dependent cell populations.