Two days before the assay, cells were plated onto poly-d-lysine–coated 96-well plates (Greiner Bio-One GmbH) and, 48 hours later, treated with DOX (1 μM) for 40 hours. Cells were washed with Dulbecco’s PBS and loaded with 3 μM Fura-2 AM (Molecular Probes) in imaging solution [5 mM KCl, 0.4 mM KH2PO4, 138 mM NaCl, 0.3 mM Na2HPO4, 2 mM CaCl2, 1 mM MgCl2, 6 mM glucose, and 20 mM Hepes (pH 7.4)] supplemented with pluronic acid (20% solution in DMSO). After incubation for 30 min at 37°C, cells were washed twice with washing buffer before being placed on the FlexStation 3 microplate reader (Molecular Devices). The Fura-2 signal was acquired at 510 nm by switching the excitation wavelength between 340 and 380 nm. [Ca2+]i was expressed as a 340/380-nm ratio, and values were normalized to the basal 340/380-nm ratio recorded during 30 s before perfusion of the drug using Softmax Pro (Molecular Devices). To allow efficient coupling of Gi/o-coupled mGluRs to the PLC pathway, cells were also transfected with the chimeric Gα protein Gαqi9 (92). Concentrations of LY379268 were selected on the basis of our previous findings (47). Thus, activation of the canonical Gi/o protein–dependent pathway was tested in the presence of LY379268 (0.1 μM), but transactivation of the Gq/11 protein–dependent pathway was tested in the presence of LY379268 (10 and 100 μM).

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