The 86Rb+ efflux data above suggest that α5 subunits transcribed in the MHb are incorporated into presynaptic α5* nAChRs in the IPN where they may regulate neurotransmitter release. Acetylcholine and glutamate are the major neurotransmitters produced by MHb neurons innervating the IPN39, and presynaptic α5* nAChRs are thought to regulate glutamate but not acetylcholine release in IPN29,40,41 Interestingly, glutamatergic transmission at the MHb-IPN synapse is increased in response to nicotine concentrations likely achieved in the brains of smokers42. We therefore hypothesized that deficient α5* nAChR signaling in the habenulo-interpeduncular tract may decrease nicotine-evoked glutamatergic transmission in the IPN and thereby attenuate a negative motivational signal that limits its intake. Consistent with this hypothesis, an aversive higher dose of nicotine (1.5 mg kg−1)43, but not a rewarding lower dose (0.5 mg kg−1)43, robustly activated the IPN in wildtype mice, reflected in increased Fos immunoreactivity (Fig. 4a,b). This effect of the high nicotine dose was almost completely abolished in the knockout mice. Wildtype and α5 knockout mice displayed similar Fos immunoreactivity in the ventromedial hypothalamus (Supplementary Fig. 12), a region in which
