Adenosine (ADO) controls neuronal excitability in memory and sleep. It is believed that ADO level is regulated by degradation of extracellular ATP directly released from neurons and glia. However, it is still controversial how extracellular ADO is regulated. One of the problems is lack of the methods to evaluate its spatiotemporal dynamics. To understand how ADO is produced in the brain, we established a novel method to visualize extracellular ADO. We first used an adeno-associated virus to express GRABAdo, a genetically encoded ADO sensor, in hippocampal astrocytes. In acute hippocampal slices, we applied the Schaffer collateral electrical stimulation (30-900 pulses, 40 Hz, 0.1 mA) to induce ADO in response to neuronal activities. The ADO level was globally increased in a stimulus-dependent manner and was gradually reduced to baseline following termination of the electrical stimulation. Since microglia expresses enzymes to metabolize ATP, we investigated the role of microglia in the ADO elevation by microglia depletion in mice fed a diet containing PLX 5622. Surprisingly, microglia depletion completely abolished the increases of the ADO. The data suggest that microglia may be a major regulator of extracellular ATP metabolism to produce ADO. The method can be useful to study neuron-glia interactions via ADO pathway.