Calcium flux analysis

For calcium flux analysis, hippocampal cultures from fetal macaque were prepared as described above, seeded onto 110-μm-thick coverslips, and cultured as above. Cells were washed with PBS and loaded with 5-μM Fura-2 (Molecular Probes, Eugene, OR) for 1 h in a dark chamber at 37 °C. Cells were then washed with PBS, and DMEM/1% FCS was added to the cultures. Neurons were kept at 37 °C until analyzed for calcium flux responses (up to 1 h). For calcium flux analysis, coverslips were placed into a 37 °C warming chamber and examined under an inverted microscope connected to a spectrofluorometer. Fetal macaque neurons with infection of the junction site-targeting siRNAs against circGRIA1 or the mismatched junction site-targeting siRNA-control, respectively, were tested for response to CTZ. Neurons were stimulated with 25-μM glutamate before exposure to 100-μM CTZ. Calcium flux tracings were analyzed for the maximum increase in intracellular calcium according to the formula [Ca]_i = k_d [(R − R_min/R_max − R)], assuming a K_d of 224 nM, and R is the ratio of fluorescence at 340 and 380 nM. Calcium concentrations are expressed as the mean ± S.D., and all data were presented as the relative ratio of fluorescence at an emission frequency of 510 nm and excitation frequencies of 380 nm and are representative of three separate experiment per treatment with five different neuronal preparations.