GABA uptake-dependent Ca²⁺ signaling in developing olfactory bulb astrocytes

We studied GABAergic signaling in astrocytes of olfactory bulb slices using confocal Ca²⁺ imaging and two-photon Na⁺ imaging. GABA evoked Ca²⁺ transients in astrocytes that persisted in the presence of GABAA and GABAB receptor antagonists, but were suppressed by inhibition of GABA uptake by SNAP 5114. Withdrawal of external Ca²⁺ blocked GABA-induced Ca²⁺ transients, and depletion of Ca²⁺ stores with cyclopiazonic acid reduced Ca²⁺ transients by approximately 90%. This indicates that the Ca²⁺ transients depend on external Ca²⁺, but are mainly mediated by intracellular Ca²⁺ release, conforming with Ca²⁺-induced Ca²⁺ release. Inhibition of ryanodine receptors did not affect GABA-induced Ca²⁺ transients, whereas the InsP₃ receptor blocker 2-APB inhibited the Ca²⁺ transients. GABA also induced Na⁺ increases in astrocytes, potentially reducing Na⁺/Ca²⁺ exchange. To test whether reduction of Na⁺/Ca²⁺ exchange induces Ca²⁺ signaling, we inhibited Na⁺/Ca²⁺ exchange with KB-R7943, which mimicked GABA-induced Ca²⁺ transients. Endogenous GABA release from neurons, activated by stimulation of afferent axons or NMDA application, also triggered Ca²⁺ transients in astrocytes. The significance of GABAergic Ca²⁺ signaling in astrocytes for control of blood flow is demonstrated by SNAP 5114-sensitive constriction of blood vessels accompanying GABA uptake. The results suggest that GABAergic signaling is composed of GABA uptake-mediated Na⁺ rises that reduce Na⁺/Ca²⁺ exchange, thereby leading to a Ca²⁺ increase sufficient to trigger Ca²⁺-induced Ca²⁺ release via InsP₃ receptors. Hence, GABA transporters not only remove GABA from the extracellular space, but may also contribute to intracellular signaling and astrocyte function, such as control of blood flow.