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BackgroundMicroglia play a critical role in neurodegenerative disorders, such as Alzheimer's disease, where alterations in microglial function may result in pathogenic amyloid-β (Aβ) accumulation, chronic neuroinflammation, and deleterious effects on neuronal function. However, studying these complex factors in vivo, where numerous confounding processes exist, is challenging, and until recently, in vitro models have not allowed sustained culture of critical cell types in the same culture. ObjectiveWe employed a rat primary tri-culture (neurons, astrocytes, and microglia) model and compared it to co-culture (neurons and astrocytes) and mono-culture (microglia) to study microglial function (i.e., motility and Aβ clearance) and proteomic response to exogenous Aβ. MethodsThe cultures were exposed to fluorescently-labeled Aβ (FITC-Aβ) particles for varying durations. Epifluorescence microscopy images were analyzed to quantify the number of FITC-Aβ particles and assess cytomorphological features. Cytokine profiles from conditioned media were obtained. Live-cell imaging was employed to extract microglia motility parameters. ResultsFITC-Aβ particles were more effectively cleared in the tri-culture compared to the co-culture. This was attributed to microglia engulfing FITC-Aβ particles, as confirmed via epifluorescence and confocal microscopy. FITC-Aβ treatment significantly increased microglia size, but had no significant effect on neuronal surface coverage or astrocyte size. Upon FITC-Aβ treatment, there was a significant increase in proinflammatory cytokines in tri-culture, but not in co-culture. Aβ treatment altered microglia motility evident as a swarming-like motion. ConclusionsThe results suggest that neuron-astrocyte-microglia interactions influence microglia function and highlight the utility of the tri-culture model for studies of neuroinflammation, neurodegeneration, and cell-cell communication.