Cortical astrocytes flexibly encode reward contingencies and shape conditioned behavior

Learned associations between environmental cues and reward drive motivated behavior, yet how specific cell types support this process remains unclear. Using longitudinal two-photon calcium imaging, we tracked dorsal medial prefrontal cortical astrocytes throughout the acquisition, expression, and reversal of Pavlovian sucrose conditioning. As learning progressed, astrocytes exhibited time-locked, spatially coordinated calcium signals that differentiated correct behavioral action from mistakes, evolving from broad outcome encoding to selective representation of responses associated with the reward-conditioned stimulus. Omission testing revealed that prefrontal astrocytes preferentially respond to the cue-reward association, rather than the conditioned stimulus or reward alone. When reward contingencies were reversed, astrocytic activity rapidly adapted to track the new cue-reward association and encode updated and outdated motivated behavioral actions. Finally, astrocytic ablation attenuated motivated behavior during initial associative learning and prevented persistence of conditioned reward seeking when reward contingencies were updated or unpredictable. These findings reveal prefrontal astrocytes are functionally plastic elements that regulate reward-seeking behavior across associative learning. TeaserPrefrontal astrocytes flexibly encode the cue-reward associations that drive conditioned reward-seeking behavior.