Microglia, the major immune cells of the brain, play a critical role in neuroinflammation caused by neurodegenerative diseases. Their functional properties in the diseased brain remain, however, unclear. Using in vivo two-photon imaging, we analyzed properties of microglia in the cortex of two different mouse models (APP23PS45/CX₃CR1^GFP/+ and APPPS1/Iba1-GFP) of Alzheimer's disease (AD). In the AD brain there were three morphologically different types of microglia: ramified, hypertrophic and amoeboid. Hypertrophic/amoeboid microglia resided in the immediate vicinity of amyloid plaques whereas ramified microglia predominantly dwelled in plaque-free areas. When challenged with 5 mM ATP applied via a patch-like pipette to mimic tissue injury (Davalos et al., 2005), all ramified microglia extended their processes towards the ATP-containing pipette, but only a fraction (27%) of amoeboid cells did so. The velocity of process extension was higher in AD compared to wildtype (WT) mice (2.27 vs. 1.79 mm/min, p=0.03, Mann-Whitney test), whereas the diameter of the spherical containment surrounding the pipette was smaller (7.06 vs. 8.15 mm, p=0.01). Next, we studied intracellular Ca²⁺ signaling of microglia. In juvenile WT mice only 22% of microglia were spontaneously active (Eichhoff et al., 2011). This fraction increased to 33% in 8–15-month-old WT and increased further to 75% in AD mice. The AD-mediated microglial hyperactivity was not caused by surrounding hyperactive neurons (Busche et al., 2008), because it persisted in the presence of TTX. Thus, our data reveal an AD-mediated impairment of functional properties of microglia likely causing a deficient response to pathological insults.