Next, we developed and validated a lentivirus vector to deliver a short-hairpin interfering RNA against the α5 nAChR subunit (Lenti-α5-shRNA; Supplementary Fig. 9). We then microinjected the Lenti-α5-shRNA vector into the MHb of rats to knockdown habenulo-interpeduncular α5* nAChRs (Supplementary Fig. 10). As expected, Lenti-Control rats responded for nicotine according to an inverted U-shaped dose-response curve (Fig. 3a). There was a dramatic increase in nicotine consumption across the dose-response curve in the Lenti-α-shRNA rats that was most apparent at high unit doses (Fig. 3a). When total nicotine intake at each dose was calculated, we found that Lenti-Control rats titrated their responding to consume ~0.75–1 mg kg−1 nicotine per session (Supplementary Fig. 10). In contrast, knockdown rats showed little evidence of titration and continued to increase their consumption as the unit dose increased. We obtained similar effects on nicotine intake using a second lentivirus vector that expressed an shRNA targeting a different portion of α5 subunit mRNA (Supplementary Fig. 11). Overall, these findings in rats recapitulate those in the α5 knockout mice and confirm that α5* nAChRs in the habenulo-interpeduncular pathway regulate levels of nicotine intake across species.
