Using radiolabeled rubidium (86Rb+) efflux as a functional measure of nAChR signaling, we found that acetylcholine-evoked 86Rb+ efflux was dramatically attenuated in synaptosomes prepared from the habenula and IPN, but not the cortex or hippocampus, of a separate cohort of knockout versus wildtype mice (Supplementary Fig. 7). Consistent with a recent report, 86Rb+ efflux was also attenuated in synaptosomes from the thalamus of knockout mice35 (Supplementary Fig. 7). Injections of lenti-CHRNA5 into MHb attenuated the deficits in 86Rb+ efflux in IPN, but not in MHb or thalamus, of knockout mice (Supplementary Fig. 8). These findings demonstrate that α5* nAChRs play a critical role in regulating nAChR transmission in the habenulo-interpeduncular tract, and confirm that the Lenti-CHRNA5 vector rescues not only expression, but also function, of α5* nAChRs in the habenulo-interpeduncular pathway. These data also reveal three additional insights: First, α5 subunits produced in MHb are predominately incorporated into α5* nAChRs expressed presynaptically on afferents to IPN. Second, injections of the Lenti-CHRNA5 vector into MHb rescued local α5 subunit mRNA expression, but not deficits in MHb 86Rb+ efflux. This suggests that
