Chronic nicotine upregulates nicotinic acetylcholine receptors (nAChR) throughout the brain, and reducing their activity may promote somatic and affective states that lead to nicotineseeking. nAChRs are functionally upregulated in animal models using passive nicotine administration, but whether/how it occurs in response to volitional nicotine intake is unknown. The distinction is critical, as drug self-administration (SA) can induce neurotransmission and cellular excitability changes that passive drug administration does not. In this study, we probed the question of whether medial habenula (MHb) nAChRs are functionally augmented by nicotine self-administration. Male rats were implanted with an indwelling jugular catheter and trained to nose poke for nicotine infusions. A saline SA group controlled for non-specific responding and nicotine-associated visual cues. Using patch clamp whole cell recordings and local application of acetylcholine, we observed robust functional enhancement of nAChRs in MHb neurons from rats with a history of nicotine SA. To determine whether upregulated receptors are generally enhanced or directed to specific cellular compartments, we imaged neurons during recordings using 2-photon laser scanning microscopy. nAChR activity at the cell soma and on proximal and distal dendrites was examined by local nicotine uncaging using a photoactivatable nicotine (PA-Nic) probe and focal laser flash photolysis. Results from this experiment revealed strong nAChR enhancement at all examined cellular locations. Our study demonstrates nAChR functional enhancement by nicotine SA, confirming that volitional nicotine intake sensitizes cholinergic systems in the brain. This may be a critical plasticity change supporting nicotine addiction. 3 SIGNIFICANCE STATEMENT This study demonstrates that stable, volitional nicotine intake is sufficient in dosage and duration to robustly enhance nAChR functional activity in MHb, a brain area involved in addiction, anxiety, and fear memory. Human tobacco use, via cigarettes or electronic nicotine delivery systems (i.e., e-cigarettes), is also likely to induce such changes to nAChRs. Minimal nicotine exposure is required to induce MHb nAChR upregulation, suggesting that it occurs early in the addiction process and that the behavioral response to repeated nicotine will include a contribution from these upregulated receptors. Available drugs and current regulatory policy are not optimal for fostering tobacco cessation, highlighting the importance of identifying mechanisms that enable continued tobacco use.

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Mesolimbic nicotinic acetylcholine receptor (nAChRs) activation isnecessaryfor nicotine reinforcement behavior, but it is unknown whether selective activation of nAChRs in the dopamine (DA) reward pathway issufficientto support nicotine reinforcement. In this study, we tested the hypothesis that activation of β2-containing (β2*) nAChRs on VTA neurons is sufficient for intravenous nicotine self-administration (SA). We expressed β2 nAChR subunits with enhanced sensitivity to nicotine (referred to as β2Leu9′Ser) in the VTA of male Sprague Dawley rats, enabling very low concentrations of nicotine to selectively activate β2* nAChRs on transduced neurons. Rats expressing β2Leu9′Ser subunits acquired nicotine SA at 1.5 μg/kg/infusion, a dose too low to support acquisition in control rats. Saline substitution extinguished responding for 1.5 μg/kg/inf, verifying that this dose was reinforcing. β2Leu9′Ser nAChRs also supported acquisition at the typical training dose in rats (30 μg/kg/inf) and reducing the dose to 1.5 μg/kg/inf caused a significant increase in the rate of nicotine SA. Viral expression of β2Leu9′Ser subunits only in VTA DA neurons (via TH-Cre rats) also enabled acquisition of nicotine SA at 1.5 μg/kg/inf, and saline substitution significantly attenuated responding. Next, we examined electrically-evoked DA release in slices from β2Leu9′Ser rats with a history of nicotine SA. Single-pulse evoked DA release and DA uptake rate were reduced in β2Leu9′Ser NAc slices, but relative increases in DA following a train of stimuli were preserved. These results are the first to report that β2* nAChR activation on VTA neurons is sufficient for nicotine reinforcement in rats.Significance StatementNicotinic acetylcholine receptor (nAChR) pharmacology and neurobiology in the dopamine reward pathway is complex and it has been a challenge to identify the minimum receptor/circuit combination(s) giving rise to nicotine dependence. This study reveals that activation of β2-containing nAChRs in ventral tegmental area dopamine neurons is sufficient to support acquisition and maintenance of nicotine self-administration in rats. This work, which employs a gain-of-function approach, complements and extends prior loss-of-function experiments demonstrating the importance of these receptors in several nicotine-related behaviors. This study 1) affirms the importance of β2 nAChRs in nicotine reinforcement, and 2) provides a useful in vivo approach for developing nicotine dependence therapeutics with either nicotinic or non-nicotinic mechanisms of action.

Many tobacco smokers consume nicotine intermittently, but the underlying mechanisms and neurobiological changes associated with intermittent nicotine intake are unclear. Understanding intermittent nicotine intake is a high priority, as it could promote therapeutic strategies to attenuate tobacco consumption. We examined nicotine intake behavior and neurobiological changes in male rats that were trained to self-administer nicotine during brief (5 min) trials interspersed with longer (15 min) drug-free periods. Rats readily adapted to intermittent access (IntA) SA following acquisition on a continuous access (ContA) schedule. Probabilistic analysis of IntA nicotine SA suggested reduced nicotine loading behavior compared to ContA, and nicotine pharmacokinetic modeling revealed that rats taking nicotine intermittently may have increased intake to maintain blood levels of nicotine that are comparable to ContA SA. After IntA nicotine SA, rats exhibited an increase in unreinforced responses for nicotine-associated cues (incubation of craving) and specific alterations in the striatal proteome after 7 days without nicotine. IntA nicotine SA also induced nAChR functional upregulation in the interpeduncular nucleus (IPN), and it enhanced nicotine binding in the brain as determined via [11C]nicotine positron emission tomography. Reducing the saliency of the cue conditions during the 5 min access periods attenuated nicotine intake, but incubation of craving was preserved. Together, these results indicate that IntA conditions promote nicotine SA and nicotine seeking after a nicotine-free period.