We used AAV to deliver small hairpin RNAs to knockdown the β4 subunit in the ventral hypothalamus and sites of infusion were verified by green fluorescent protein (GFP) detection (Photograph, upper right; 3V = third ventricle; ARC = arcuate nucleus). Following recovery and knock down expression of β4 nAChRs, cytisine (1.5 mg/kg) was unable to decrease food intake contrary to the control group of animals. *** p <0.001.

POMC neurons express nicotinic acetylcholine receptors (nAChRs) and therefore respond to nicotinic drugs. In the Basal state (i.e. in the absence of nicotine), POMC neurons project to second order neurons that decrease food intake. When nicotine reaches the arcuate nucleus (facilitated by its proximity to the third ventricle), activity of POMC neurons is increased (as measured by c-fos expression and neuronal activity measured in slices) through activation of α3β4 nAChRs and subsequent activation of MC4 receptors in the paraventricular nucleus of the hypothalamus (Nicotine-activated state).

Both nicotine and cytisine dose-dependently prevented weight gain in mice treated daily for 30 days, with more pronounced effects seen at higher doses (A, all Ps < 0.001). In contrast, the non-selective nicotinic antagonist mecamylamine had no significant effect (F < 1), suggesting that nAChR antagonism alone was not sufficient for the anorexic effects of nicotinic compounds. Body fat measured by MRI was also reduced in mice treated with cytisine (1.5 mg/kg; F(1, 23) = 13.7, p = 0.006) and nicotine (0.5 mg/kg; F(1, 23) = 5.08, p = 0.03; B). Acute injection of cytisine decreased food intake after 2 hr (F(1, 18) = 100.3, p < 0.001) and this effect was still observed after 24 hr (F(1, 18) = 35.3, p < 0.001). The effect of cytisine was not blocked by the peripherally-acting nAChR antagonist hexamethonium (2 hr: F(1, 18) = 121.5, p < 0.001); 24 hr: F(1, 18) = 37.9, p < 0.001) but was blocked by mecamylamine (Fs < 1; C) indicating cytisine acts at central nAChRs to exert its anorexic effects.

Administration of nicotine (0.1 mg/kg) or cytisine (1.5 mg/kg), unlike mecamylamine (1 mg/kg), resulted in specific activation of POMC cells in the arcuate nucleus as measured by c-fos immunoreactivity (A) and electrophysiological studies further demonstrate a dose-dependent (see inset in B), reversible increase in the firing rate of identified POMC neurons in response to nicotine (B) or cytisine (C) application. Food intake was not significantly affected by nicotine or cytisine in POMC KO at two different doses (D). Furthermore, knock down of Mc4r by AAV-shRNAs in the PVN (detected by GFP fluorescence (Fig. S7)) significantly blunted the hypophagic response to nicotine over time, compared to mice injected with control virus and treated with nicotine. No signs of tolerance to nicotine were observed over 30 days, consistent with a role for MC4R signaling in the anorexic effects of nicotinic agents (E). ** p < 0.01; *** p < 0.001.