negative motivational signals in the brain34. We therefore hypothesized that nicotine-induced stimulation of α5* nAChRs in the habenulo-interpeduncular pathway triggers an inhibitory motivational signal that limits consumption of the drug. In knockout and wildtype mice that received injections of a control lentivirus expressing green-fluorescent protein (GFP; Lenti-Control) into the MHb, we again found that knockout mice self-administered far greater amounts of nicotine when a high unit dose was available (Fig. 2a), replicating the above findings. However, nicotine intake was indistinguishable in knockout versus wildtype mice after injection of a lentivirus vector (Lenti-CHRNA5) into the MHb to rescue α5 nAChR subunits in the habenulo-interpeduncular tract (Fig. 2b; Supplementary Fig. 3). GFP immunostaining to confirm MHb delivery of virus was carried out for the majority of the mice. Responding for nicotine (0, 0.1 and 0.4 mg kg−1 per infusion) in the subset of Lenti-CHRNA5-treated used for immunostaining 3.6 ± 0.83, 8.8 ± 1.4 and 4.86 ± 1.0, respectively, for wildtypes and 4.53 ± 0.85, 7.72 ± 0.68 and 4.53 ± 1.4, respectively, for knockouts. GFP immunostaining confirmed that virus-infected cells were detected almost exclusively in the habenulo-interpeduncular tract of Lenti-CHRNA5 knockout mice, with little detectable staining in other brain areas that could
