Habenular α5 nicotinic receptor subunit signalling controls nicotine intake

Abstract

Genetic variation in CHRNA5, the gene encoding the α5 nicotinic acetylcholine receptor subunit, increases vulnerability to tobacco addiction and lung cancer, but the underlying mechanisms are unknown. Here we report markedly increased nicotine intake in mice with a null mutation in Chrna5. This effect was 'rescued' in knockout mice by re-expressing α5 subunits in the medial habenula (MHb), and recapitulated in rats through α5 subunit knockdown in MHb. Remarkably, α5 subunit knockdown in MHb did not alter the rewarding effects of nicotine but abolished the inhibitory effects of higher nicotine doses on brain reward systems. The MHb extends projections almost exclusively to the interpeduncular nucleus (IPN). We found diminished IPN activation in response to nicotine in α5 knockout mice. Further, disruption of IPN signalling increased nicotine intake in rats. Our findings indicate that nicotine activates the habenulo-interpeduncular pathway through α5-containing nAChRs, triggering an inhibitory motivational signal that acts to limit nicotine intake.

Figure 1. Increased nicotine intake in α5 subunit knockout mice

(a) Data are presented as mean (± SEM) number of nicotine infusions earned across a range of nicotine doses. Two-way ANOVA: Genotype F_(1,91)=28.57, p<0.0001; Dose F_(6,91)=13.69, p<0.0001; Interaction F_(6,75)=2.55, p<0.05; n=10–11 per group. (b) Data from [a] are presented as mean (± SEM) total nicotine intake at each dose. Genotype F_(1,91)=67.98, p<0.0001; Dose F_(6,91)=39.06, p<0.0001; Interaction F_(6,791=14.25, p<0.0001.

Figure 2. “Rescue” of α5* nAChRs in MHb-IPN normalizes nicotine intake

(a) Mean (± SEM) nicotine infusions in Lenti-Control mice. Genotype F_(1,22)=7.70, p<0.05; Dose F_(2,22)=19.34, p<0.0001; Interaction F_(2,22)=3.75, p<0.05. **P<0.01 between genotypes (b) (± SEM) nicotine infusions in Lenti-CHRNA5 mice. Genotype F_(1.28)=0.17, not significant (n.s.); Dose F_(2,28)=16.05, p<0.0001; Interaction F_(2,28)=0.36, n.s.; n=6–9 per group. (c) GFP Immunostaining confirmed MHB virus delivery. Hipp, hippocampus; LHB, lateral habenula; LV lateral ventricle; MHb, medial habenula. (d) GFP-labeled cells in MHb, DAPI-counterstained in left panel, extend into the fasciculus retroflexus (Fr). (e) GFP-positive axons detected in IPN. Left panel is labeled with VAChT (red) to identify IPN.

Figure 3. α5* nAChRs in MHb-IPN tract control nicotine intake and its reward-inhibiting effects in rats

(a) Nicotine self-administration in rats injected with Lenti-Control or Lenti-α5-shRNA in the MHb. Data are presented as mean (± SEM) number of nicotine infusions earned. Lentivirus F_(1,60)=21.07, p<0.01; Dose F_(6,60)=3.84, p<0.01; Interaction F_(6,60)=1.57, n.s.;; n=5–7 per group. (b) ICSS self-stimulation thresholds in rats. Data are presented as mean (± SEM) percentage change from baseline reward threshold. Lentivirus F_(1,60)=13.23, p<0.001; Dose F_(5,60)=6.38, p<0.0001; Interaction F_(5,60)=4.19, p<0.01. *p<0.05, **p<0.01 and ***p<0.001 indicates statistically significant difference between groups; n=6–8 per group.

Figure 4. Nicotine-induced activation of IPN in mice

(a) Photomicrograph of IPN showing Fos immunoreactivity in wildtype (left panels) and α5 knockout (right panels) mice following saline (top panels), 0.5 mg kg⁻¹ nicotine (center panels), or 1.5 mg kg⁻¹ nicotine (bottom panels); n=5 per group. (b) Cell density was quantified with unbiased stereology. Data are presented as the mean (± SEM) density of Fos-immunoreactive cells (number per mm³). Genotype F_(1,24)=13.50, p<0.01; Drug F_(2,24)=21.13, p<0.0001; Interaction F_(2,24)=8.64, p<0.01.

Figure 5. Disruption of IPN or MHb signaling increases nicotine intake in rats

All data are presented as mean (± SEM) number of nicotine infusions earned. (a) Lidocaine infused into IPN increased nicotine intake in rats; **P<0.01. (b) Lidocaine into MHb increased nicotine intake in rats self-administering a high unit dose (0.12 mg kg⁻¹ per infusion); *P<0.05. (c) LY235959 infused into IPN increased nicotine intake in rats (n=9). F_(3,24)=6.08, p<0.01. *P<0.05 and **p<0.01 compared to control. (d) LY235959 (10 ng/side) into MHb increased nicotine intake in rats responding for a high unit dose (0.12 mg kg⁻¹ per infusion; n=5); *P<0.05.