Receptor blockade shows that nicotine acts via α7-nAChRs, not α4β2-nAChRs, to stabilize GluA1s on spines independent of AMPA and NMDA receptor activation. A, Time course of GluA1-SEP FRAP after 1–3 h in nicotine with and without 100 nm MLA to block α7-nAChRs (MLA vs MLA + Nic: 97 ± 5 vs 96 ± 5%, n = 12,15; p = 0.86; Ctrl vs Nic: 97 ± 5% vs 78 ± 3%, n = 14,14; p = 0.0038, WC). B, Relative GluA1-SEP enrichment on spines under conditions from A (MLA vs MLA + Nic: 1.23 ± 0.11 vs 1.17 ± 0.09; n = 16,16; p = 0.66; Ctrl vs Nic: 1.09 ± 0.10 vs 1.61 ± 0.11; n = 16,16; p = 0.0013). C, Blockade of α4β2-nAChRs with 1 μm DHβE during the nicotine treatment had no effect on GluA1-SEP mobility (DHβE vs DHβE + Nic: 93 ± 3 vs 79 ± 5%, n = 11,12; p = 0.024, WC; Ctrl vs Nic: 99 ± 3 vs 78 ± 4%, n = 12,11; p = 0.0004). D, DHβE did not prevent nicotine-induced GluA1-SEP enrichment on spines (DHβE vs DHβE + Nic: 1.03 ± 0.11 vs 1.57 ± 0.18; n = 1 2,12; p = 0.0168; Ctrl vs Nic: 0.94 ± 0.08 vs 1.57 ± 0.12; n = 12,12; p = 0.0002). E, Blockade of AMPARs and NMDARs with 50 μm APV and 20 μm NBQX, respectively, during the nicotine treatment had no effect on GluA1-SEP mobility on spines (APV + NBQX vs APV + NBQX + Nic: 98 ± 6 vs 58 ± 7%, n = 10,11; p = 0.0006; Ctrl vs Nic: 94 ± 4 vs 71 ± 5%, n = 10,11; p = 0.0017). F, APV and NBQX failed to block GluA1-SEP enrichment on spines (APV + NBQX vs APV + NBQX + Nic: 1.2 ± 0.11 vs 1.73 ± 0.21; n = 11,11; p = 0.0405, WC; Ctrl vs Nic: 1.18 ± 0.08 vs 1.67 ± 0.1; n = 11,11; p = 0.001).