Wildtype and α5 subunit knockout mice were injected subcutaneously with nicotine (0.5 or 1.5 mg/kg, free-base) or saline. The moderate dose of nicotine is known to be rewarding in these mice, reflected in the conditioning of a place preference8. The higher dose of nicotine is aversive, reflected in the induction of a conditioned taste aversion in wildtype mice9. After 2 hr, each subject was perfused and brains were removed and stored as described above. Brain sections were cut at 30μm on a cryostat and stored in 0.1M PBS with 0.01% sodium azide until processing. For Fos immunolabeling, sections were rinsed in 0.1M PBS (ph 7.4), treated with 0.3% H2O2-PBS for 15 min, rinsed in PBS, and then blocked in 10% normal goat serum and 0.5% Triton X-100 in PBS for 1 hr. Thereafter, sections were incubated in rabbit anti-cfos IgG (1:500 dilution; Abcam, Cambridge, MA) in 0.5% Triton-PBS overnight at 4°C. The following day, sections were incubated at room temperature for 2 hrs, rinsed in PBS, and then incubated in 1:300 dilution of goat anti-rabbit secondary IgG (Vector Labs) in
