± 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 potentially impact self-administration behavior (Fig. 2c,d; Supplementary Fig. 4). The remaining mice not used for immunostaining were used to verify that the Lenti-CHRNA5 virus was function. Real-time PCR verified that α5 subunit mRNA was detectable only in habenula (Supplementary Fig. 5) and IPN (Supplementary Fig. 6) of the Lenti-CHRNA5-treated knockout mice, suggesting that α5 nAChR subunit mRNA is transported from the MHb along the fasciculus retroflexus and into the IPN. Wildtype mice treated with the Lenti-CHRNA5 vector did not demonstrate increased α5 subunit mRNA above baseline levels in the habenula (Supplementary Fig. 5), suggesting that strict regulatory mechanisms control α5* nAChR expression in the MHb-IPN pathway.
