pH Sensor-Based Engulfment Assays to Measure Phagocytic Activity of Astrocytes

Astrocytes play a crucial role in eliminating unnecessary synapses, neural debris, and pathogenic proteins such as amyloid β plaque through phagocytosis. Emerging evidence suggests that the phagocytic roles of astrocytes are critical for remodeling our neural circuits during development as well as maintaining brain homeostasis in adulthood. Moreover, abnormal astrocytic phagocytosis can have direct implications for the initiation and progression of various brain disorders. However, classical methods for identifying phagocytic events in the brains have utilized immunostaining-based and/or electron microscopy-based imaging analysis, which are very labor-intensive and often produce subjective results due to the inaccuracy in isolating the engulfment versus attachment to the plasma membrane of phagocytes. To overcome this problem, we have developed efficient in vitro and in vivo engulfment assays based on pH sensors, where only engulfed materials in astrocytes can be isolated by the changes in fluorescent signals, providing valuable tools for measuring phagocytic capacity and kinetics of astrocytes. In this chapter, we will explain and discuss two of our assays: (1) an in vitro engulfment assay using purified astrocytes and synaptosomes and (2) a mCherry-eGFP reporter system for monitoring in vivo synapse phagocytosis in the brains. Using these in vitro and in vivo assays, we have been able to accurately quantify the amount of synapses engulfed by multiple glial cells, including astrocytes, microglia, and oligodendrocyte precursor cells, revealing distinct roles and mechanisms of glial phagocytosis in eliminating specific synapse populations from defined neural circuits. These advanced assays provide accurate and efficient means of studying glial phagocytosis, contributing to a better understanding of neural circuit remodeling and the maintenance of brain homeostasis.