<p>Intracellular calcium ion (Ca²⁺i) is one of the most important cation that controls several cellular functions, and thus there were already huge number of literature about Ca²⁺i imaging in various cells. Although it is believed that Ca²⁺i increase is accompanied by extracellular Ca²⁺(Ca²⁺o) decrease, the kinetics of Ca²⁺o decrease remain unknown because of the limited imaging options. To overcome this limitation, we developed a Ca²⁺ image sensor (CIS) that is highly selective to Ca²⁺ but not to other cations within wide dynamic range (from 100 mM to 100 mM). We used CIS for the imaging of Ca²⁺o in acute hippocampal slices. Stimulation with glutamate (Glu) decreased Ca²⁺o to around 200 nM within 3 s, which returned to the baseline level (2 mM) with slow kinetics (5 min). Glu stimulates both neurons and glial cells to evoke Ca²⁺i elevation, thereby reducing Ca²⁺o. Thus, we tested NMDA to selectively stimulate NMDA receptor in neurons. NMDA mimicked Glu-evoked Ca²⁺o decrease. Interestingly, Ca²⁺o decrease was initiated at hippocampal CA1-2, before spreading along the pyramidal layers. Glutamate- and NMDA-evoked Ca²⁺o decreases were inhibited by a NMDA receptor antagonist D-APV, suggesting involvement of neuronal NMDA receptors in decrease in Ca²⁺o. So far, many scientists neglected Ca²⁺o, but the CIS would tell us its importance for understanding brain functions.</p>