Disruption of lipid rafts decreases mGlu1 receptor-induced Ca2+transient and receptor localization to lipid rafts in hippocampal neurons. (A) A representative trace of Fura-2 Ca2+ imaging is shown. Cells loaded with Fura-2/AM in NT solution for 30 min were stimulated with 50 μM DHPG during 60s (arrows) in the presence of MPEP (10 μM) to block mGlu5 receptor activation. DHPG was also given after treatment of mβCD (2 mg/ml for 300 sec) for cholesterol depletion and mβCD/cholesterol complex for cholesterol replenishment (10 μM for 300 sec) as well. Gray and black bars represent treatment of mβCD and mβCD/cholesterol complex. Population data are shown in right panel. n=33. (B) Representative field images from Ca2+ imaging of A. Images are presented, left to right: Baseline, imaging field of baseline. DHPG, at the peak ratio by applying DHPG. mβCD and mβCD/Cholesterol, the peak in the presence of mβCD and mβCD/Cholesterol. Cells analyzed are numbered in the baseline image. Scale bar for Fura-2 ratio is shown left. (C) Confocal images visualizing co-localization of mGlu1α receptor (red) and lipid rafts (green) following mβCD and mβCD/cholesterol complex. White boxes in the upper figures are enlarged in lower figures. Overlaying areas (yellow) are designated with asterisks, implying co-localization of mGlu1α receptor to CTX-Alexa 488. Spines are indicated with white arrow heads. Co-localization scores are presented in right panel. n = 11 (Control), 12 (mβCD), and 15 (mβCD/cholesterol complex). Scale bar = 10 μm. The data is shown as mean ± SEM. ANOVA followed by post hoc test, n.s., non-significant, *, P < 0.05, **, P < 0.001.