β2 nAChR Activation on VTA DA Neurons Is Sufficient for Nicotine Reinforcement in Rats

Abstract Mesolimbic nicotinic acetylcholine receptor (nAChRs) activation is necessary for nicotine reinforcement behavior, but it is unknown whether selective activation of nAChRs in the dopamine (DA) reward pathway is sufficient to 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 (SD) 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.


Introduction
Nicotine, the key psychoactive compound in tobacco products, is paradoxical; it has potent psychostimulant properties but is nevertheless a weak reinforcer. A variety of behavioral, pharmacological, genetic, and circuit-based approaches have been used to examine this issue with the goal of understanding the mechanistic basis for nicotine dependence/addiction. Identification of nicotinic acetylcholine receptor (nAChR) subtypes responsible for nicotine's reinforcing and dependence-producing property has been of great interest, as accomplishing this task may lead to new drugs for improved smoking cessation outcomes. Most current knowledge comes from loss-of-function experiments. For example, Corrigall used a pharmacological approach to demonstrate that b 2* nAChR activity is necessary for nicotine self-administration (SA) in a rat model (Corrigall et al., 1994). Using knock-out (KO) mice, Picciotto and colleagues identified the b 2 nAChR subunit as necessary for the transfer of operant responding for cocaine to responding for nicotine (Picciotto et al., 1998). Using an active versus yoked paradigm with mice having only a single nicotine IVSA session, Changeux and colleagues identified the b 2 nAChR subunit as necessary for nicotine self-administration (Pons et al., 2008). These loss-of-function studies indicate the importance of b 2 nAChR subunits in nicotine reward and self-administration behavior, but they do not address the question of b 2 sufficiency.
nAChR gain-of-function approaches, involving expression of nAChR subunits with increased sensitivity to agonist, complement loss-of-function experiments and provide a useful and alternative method to examine the molecular basis for nicotine-related behaviors . Early molecular pharmacology work on nAChRs that focused on understanding the gating mechanism employed mutagenesis of a nearly universally conserved leucine residue (the 99 Leucine) in the TM2 a-helix, resulting in a slowing/reduction in desensitization and an increase in sensitivity (Revah et al., 1991;Bertrand et al., 1992;Yakel et al., 1993;Filatov and White, 1995). This effect is thought to arise from stabilization of the open state, and a serine substitution (used in this study) strongly induces the effect whereas alanine or hydrophobic substitutions are more modest (Revah et al., 1991;Bertrand et al., 1992;Yakel et al., 1993).
A key discovery related to the present work is that Leu99 mutant subunits incorporate into pentameric receptors with either wild-type (WT) or other Leu99 mutant subunits, and receptor sensitivity (or hypersensitivity) is directly related to the number of Leu99 mutant subunits in each pentamer (Labarca et al., 1995). Leu99 mutant subunits can therefore be expressed on a background of WT subunits, making them useful for in vivo experiments in behaving animals.
nAChR subunits modified at the Leu99 position have been useful for understanding nicotine-related behaviors and neurobiology. Conditioned place preference experiments with knock-in mice expressing Leu99Ala a4 subunits demonstrated that activation of a4b 2 nAChRs is sufficient for nicotine reward-like behavior (Tapper et al., 2004). Studies with transgenic mice expressing Leu99Ser a6 subunits revealed a myriad of behavioral/pharmacological functions for a6b 2 nAChRs (Drenan et al., 2008(Drenan et al., , 2010Cohen et al., 2012;Engle et al., 2013Engle et al., , 2015Powers et al., 2013;Wang et al., 2014;Berry et al., 2015;Wieskopf et al., 2015). However, nicotine self-administration is difficult to establish in mice, so the Leu99 approach has not been fully used to make key insights into pharmacological/cellular/ circuit mechanisms of nicotine reinforcement.
To address this gap in the field, we employed the Leu99 approach to answer the following questions: is selective activation of VTA b 2 nAChRs sufficient for acquisition of nicotine self-administration and if so, is b 2 nAChR activation reinforcing? We designed and produced an adenoassociated virus (AAV) for expression of rat b 2 nAChR subunits with a Leu99 to Serine substitution. This vector was delivered via stereotaxic surgery to the brain of Sprague Dawley (SD) or transgenic TH-Cre Long-Evans rats, triggering expression of hypersensitive b 2 nAChRs for subsequent intravenous nicotine self-administration experiments and physiology studies.

Molecular biology
We conceived of and designed the AAV5.2-hSyn-DIO-Chrnb2Leu273Ser-P2A-GFP (b 2 Leu99Ser) vector, which was then prepared by Virovek AAV5-hSyn-mCherry-Cre was obtained from the University of North Carolina Vector Core Facility. Rat a4 and b 2 nAChR subunits were cloned into the pGEMhe vector (Marotta et al., 2014). The Leu99Ser mutation was introduced into the b 2 subunit vector via the QuikChange protocol (Stratagene). The a4 and b 2 circular DNA vectors were linearized using the Sda I (Sbf I) enzyme (Thermo Fischer Scientific). Qiaquick PCR Purification kit (QIAGEN) was used to purify the linearized DNA. mRNA was transcribed in vitro from the linearized DNA using the mMessage Machine T7 Transcription kit (Ambion). The resultant mRNA was isolated using the RNeasy RNA Purification kit (QIAGEN).

Animals
All experimental protocols involving vertebrates were reviewed and approved by the Wake Forest University School of Medicine Institutional Animal Care and Use Committee. Procedures also followed the guidelines for the care and use of animals provided by the National Institutes of Health Office of Laboratory Animal Welfare. All efforts were made to minimize animal distress and suffering during experimental procedures, including during the use of anesthesia. A total of n = 85 male SD rats (Envigo) were used. SD rats were ;300 g (approximately eight weeks old) when they arrived at our facility. Rats were housed at 22°C on a reverse 12/12 h light/dark cycle (4 P.M. lights on, 4 A.M. lights off). Transgenic Long-Evans TH::Cre rats (Witten et al., 2010(Witten et al., , 2011 were obtained from Rat Resource and Research Center (RRRC). A breeding colony was established for TH-Cre rats by breeding heterozygous TH-Cre males with WT Long-Evans females (obtained from Envigo). All progeny from breeding pairs were genotyped (Transnetyx), and nontransgenic littermates were used as controls. A total of n = 20 male TH-Cre (n = 14) or non-Tg littermate (n = 6) rats were used for experiments. Xenopus laevis oocytes were purchased from EcoCyte Bioscience.

Apparati
Rats were trained in Med Associates operant chambers (interior dimensions, in inches: 11.9 Â 9.4 Â 11.3, catalog #MED-007-CT-B1) located within sound-attenuating cabinets. The SA system was housed in a dedicated room within the same laboratory suite as the rat's housing room. A PC computer was used to control the SA system via Med PC IV software. Each chamber had transparent plastic walls, a stainless-steel grid floor, and was equipped on the right-side wall with two nose pokes (2.4 inches from grid floor to nose poke center) which flanked a pellet receptacle coupled to a pellet dispenser. A white stimulus light was located above each nose poke, and a house light was located at the top of the chamber on the left-side wall. During food and drug SA sessions, nose pokes on the active nose poke activated either the pellet dispenser or an infusion pump, respectively. Nose pokes on the inactive nose poke had no consequence. For intravenous drug infusions, each rat's catheter was connected to a liquid swivel via polyethylene tubing protected by a metal spring. The liquid swivel was connected to a 10-ml syringe loaded onto the syringe pump.

Operant food training
Approximately one week after arrival, pair-housed rats were food-restricted for several days to enhance their participation in operant responding. Rats were fed standard chow (LabDiet Prolab RMH 3000 5P00, catalog #0001495; 40 g per cage) once per day at least 1 h after finishing testing. Water was available ad libitum except during operant behavioral sessions. Food training sessions were 1 h in duration, and rats were trained to nose poke for food pellets (45 mg; Bio-Serv Dustless Precision Pellets, catalog #F0021) on the same nose poke that would subsequently be paired with drug infusions in nicotine IVSA sessions. A fixed ratio 1 (FR1; no timeout) schedule was used for food training; no visual cues (stimulus light, house light) were illuminated during the session and rats could earn a maximum of 75 food pellets during the 1-h session. Once each rat successfully earned at least 50 pellets with at least a 2:1 preference for the active nose poke over the inactive nose poke, no further food training was conducted. Rats met this criterion on average between 3 and 5 d. Food training was used to shorten the time required for rats to acquire nicotine SA, though it is not required.

Stereotaxic surgery
After arrival at our facility, rats were anesthetized with isoflurane (3% induction, 2-5% maintenance) and were introduced into a stereotaxic rat frame. Rats received a bilateral injection into the VTA using coordinates from bregma (SD rats: AP: À5.25, ML: 61.00, DV: À8.00; TH::Cre rats: AP: À5.40, ML: 60.70, DV: À8.20). Coordinates were derived from "Brain Maps 4.0" (Larry Swanson; University of Southern California; Swanson, 2018). Virus was infused using a 22-gauge Hamilton injection syringe at a rate of 100 nl/min and the injection needle was left in place for 5 min after each injection before gradually being retracted. Rats were then given 6-7 d for recovery.

Indwelling jugular catheter surgery
After acquiring food operant responding, rats were anesthetized with isoflurane (3% induction, 2-3% maintenance) and implanted with indwelling jugular catheters (Instech, catalog #C30PU-RJV1402). Meloxicam (2 mg/ kg) was administered postoperatively to relieve pain and reduce inflammation. Rats were singly housed following surgery and throughout all SA procedures. Rats were allowed 7 d for recovery from surgery, and catheters were flushed several times during this recovery period with heparin sodium dissolved in sterile saline.

Intravenous drug self-administration
After recovery from catheter surgery, rats were allowed to self-administer saline or nicotine (dose variable, as described in Results) in a volume of 0.035 ml over 2 s during 2-h SA sessions, Monday through Friday (no SA sessions occurred on weekends). (À)-Nicotine hydrogen tartrate salt (Glentham Life Sciences) was dissolved in sterile saline, and the pH was adjusted to 7.4. Infusions, delivered by an infusion pump, were triggered by one nose poke response on the active nose poke. Infusions (2-s duration) were simultaneously paired with illumination of the stimulus light over the active nose poke for 3 s. An active nose poke response that resulted in an infusion extinguished the house light for a 20-s timeout period (TO-20s), during which responding was recorded but had no consequence. Responses on the inactive nose poke were recorded but had no scheduled consequence. At the end of the session, the house light was extinguished and responding had no consequences. Rats were removed from the training chambers as soon as possible after the end of the 2-h session and were rationed to 20 g of standard lab chow at least 1 h after finishing their sessions. Modified chow availability was used throughout SA and a range of 85-90% of free-feeding body weight was maintained. SD rats were allowed to selfadminister nicotine (30 mg/kg/inf or 1.5 mg/kg/inf) for 10 d on an FR1/TO-20s schedule of reinforcement. For rats receiving 30 mg/kg/inf, n = 2 of 8 injected SD b 2Leu99Ser::Cre(À) rats, n = 2 of 10 injected SD b 2Leu99Ser::Cre(1) rat, and n = 0 of 7 control SD rats failed acquisition. Acquisition failure could be triggered by the occurrence of any one of the following: (1) ,10 nicotine infusions were earned within the 2-h session for two or more consecutive sessions, (2) the ratio of active to inactive nose pokes was ,2.0 for three or more consecutive sessions, and (3) a drop of 75% or greater in responding on the active nose poke occurred during sessions #6-10 of acquisition. For SD b 2Leu99Ser::Cre(1) rats receiving 1.5 mg/kg/inf nicotine, n = 8 of 36 injected rats failed acquisition because of a failure to maintain an average of 11.1 infusions across SA days 6-10. This value is based on our prior work with saline SA, where naive SD rats typically average this many saline self-infusions across SA days 6-10 under the identical operant/cue conditions. Using this same metric, n = 7 of 8 injected SD b 2Leu99Ser:: Cre(À) rats receiving 1.5 mg/kg/inf nicotine failed to acquire SA, and n = 9 (of nine total) SD control rats receiving 1.5 mg/ kg/inf nicotine failed to acquire SA. For Long-Evans TH-Cre rat nicotine (at 1.5 mg/kg/inf) SA acquisition, we calculated the average # of infusions across SA days 11-17 in n = 6 non-Tg littermate rats injected with b 2Leu99Ser vectors. This value, plus 2Â the SD (equal to approximately six infusions), was used as a cutoff to determine acquisition in n = 11 TH-Cre rats injected with b 2Leu99Ser vectors. Based on this criterion, n = 1 of 11 total injected TH-Cre rats failed acquisition because of an average # of infusions across days 11-17 below 6.

Ex vivo fast scan cyclic voltammetry
On the morning of recordings, rats were deeply anesthetized with isoflurane and decapitated. The brain was removed and immersed in oxygenated artificial CSF (aCSF) containing (in mM): 126 NaCl, 2.5 KCl, 1.2 NaH 2 PO 4 , 2.4 CaCl 2 , 1.2 MgCl 2 , 25 NaHCO 3 , 11 glucose, 0.4 l-ascorbic acid. A vibrating tissue slicer (Leica VT1200S, Leica Biosystems, Wetzler, Germany) was used to prepare 400 mm thick coronal brain slices containing the NAc core (NAcc). Slices were transferred to a recording chamber and submerged in a bath of oxygenated aCSF (32°C) perfused at a rate of 1 ml/min. A carbon-fiber microelectrode (CFE; 150-200 mM length, 7-mm radius) and bipolar stimulating electrode were placed in the NAc core. Endogenous dopamine (DA) release was electrically evoked by a single pulse (350 mA, 7.5 V, 4 ms, monophasic) applied to the slice at 5 min intervals. Extracellular dopamine was measured by applying a triangular waveform (À0.4 to 1 1.2 to À0.4 V vs Ag/AgCl, 400 V/s) to the CFE. Baseline dopamine release and uptake kinetics were considered stable after an hour of slice acclimation and when the dopamine signal was stable for at least three successive collections. The effects of two different stimulation patterns on dopamine release were tested. A stimulation intensity relation was collected by using a single pulse stimulation at varying intensities, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 V with an interstimulus interval of 60 s. A frequency-response relation was obtained using four-pulse stimulations at 3, 10, 30, and 100 Hz with 5min interstimulus intervals. Dopamine response was determined following a concentration response curve using bath application of nicotine (1 to 100 nM), with doses increasing on a half-log scale. Once a stable dopamine signal was collected at the baseline and each dose, a single four-pulse, 30-Hz stimulation was applied for a single file collection. Demon Voltammetry and Analysis software (freely available from Wake Forest University School of Medicine) was used to analyze all FSCV data (Yorgason et al., 2011). Calibration factors for individual recording electrodes were determined using a multiple linear regression model of preestablished background currents and resulting dopamine sensitivity. The calibration factor of each electrode was used to convert the electrical current measured during experiments to dopamine concentration. Michaelis-Menten modeling was used to determine the concentration of dopamine released and the maximal rate of uptake (V max ) following electrical stimulation.

Two-electrode voltage clamp electrophysiology
ND96 Ca 21 free buffer (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES at pH 7.5) was used to generate a 1 M ACh stock solution. A series of ;3-fold concentration steps were made and serially diluted for several orders of magnitude, totaling 12-15 doses for electrophysiology studies. A 1:2 mass ratio of a4:b 2 mRNA were mixed to deliver 10 ng of total mRNA in a 50 nl injection volume. Oocytes were injected and then incubated at 18°C in ND961 medium with 5% horse serum for 24 h. All electrophysiology recordings were performed in two-electrode voltage clamp mode on an OpusXpress 6000A (Molecular Devices). The running buffer was ND96 Ca 21 free and holding potentials were -60 mV for all experiments. 1 ml ACh drug applications were applied over 15 s followed by 3 min of buffer (3 ml/min). Doses were applied from lowest to highest concentrations. Data were sampled at 50 Hz and then low-passed filtered at 5 Hz. Maximum current values were recorded for ACh doses. These were normalized, averaged, and fit to a one Hill equation model to calculate EC 50 and Hill coefficient values. Error bars represent the SEM.

Patch clamp electrophysiology
Electrophysiology experiments were conducted using a Nikon Eclipse FN-1 upright microscope equipped with a 40Â (0.8 NA) water-dipping (3.3-mm working distance) objective. Neurons in the VTA were targeted for recording. Neurons within brain slices were first visualized with infrared or visible differential interference contrast (DIC) optics. A computer running pCLAMP 10 software was used to acquire whole-cell recordings along with a Multiclamp 700B amplifier and a Digidata 1550A A/D converter (all from Molecular Devices Inc.). Data were sampled at 10 kHz and low pass filtered at 1 kHz. Immediately before giga seal formation, the junction potential between the patch pipette and the superfusion medium was nulled. Series resistance was uncompensated. To record physiological events following local application of drugs, a drug-filled pipette was moved to within 20-40 mm of the recorded neuron using a second micromanipulator. A Picospritzer (General Valve) dispensed drug (dissolved in recording solution) onto the recorded neuron via a pressure ejection. Pipette location relative to the recorded cell, along with ejection pressure, were held constant throughout the recording. Ejection duration was varied to enable collection of quasi-concentration response curves.

Nicotine pharmacokinetic modeling
Nicotine pharmacokinetic simulations in rats were conducted using MATLAB and SimBiology (MathWorks). A two-compartment model was built, simulating a central (blood, plasma, CSF, etc.) and peripheral (fat, muscle, and other poorly perfused tissues) compartment. Hereafter, "plasma" denotes the central compartment. Model parameters were selected based on previous studies (Kyerematen et al., 1988;Shivange et al., 2019). Key model parameters include: central compartment capacity (5 l/kg), elimination rate constant (0.8), forward rate constant (central-.peripheral; 1.5), reverse rate constant (peripheral-.central; 1.2), and clearance rate constant (1.4). Limitations include, but are not limited to, the following: (1) a standard 350-g rat was modeled, and (2) variability in nicotine metabolism across animals was not accounted for. Model parameters were first validated by comparing rat plasma nicotine kinetics following an arterial bolus dose (Kyerematen et al., 1988) to the predicted nicotine kinetics predicted by the model after simulating the same bolus dose into the central compartment. Moreover, the peak predicted plasma [nicotine] levels predicted by the model agree with other reports that measured rat nicotine levels in plasma after intravenous dosing (Craig et al., 2014). To model predicted plasma nicotine levels in our experimental rats, the model was enabled to accept infusion time vectors from SA sessions. The model returned a predicted [nicotine] versus time profile, which was used to calculate key metrics such as peak predicted plasma [nicotine] and predicted plasma area under the curve (AUC) for specified time periods after the start of the SA session. Predicted steady-state plasma [nicotine] levels (10-50 ng/ml) in our experimental rats during nicotine SA sessions were similar to plasma nicotine levels measured in human smokers during unrestricted cigarette smoking (Benowitz et al., 1983) and during regular, intermittent cigarette smoking (Isaac and Rand, 1972;Benowitz et al., 1982). In particular, we observed peaks and troughs in the predicted plasma nicotine versus time profile that were reminiscent of data from humans during intermittent cigarette smoking (Isaac and Rand, 1972).

Experimental design and statistical analysis
SA data files, produced by Med Associates MedPC IV software, were analyzed with custom scripts written in MATLAB and/or GraphPad Prism 9. All sample sizes and results from statistical analyses for Figures 1-6 are presented in Table 1. Patch clamp and two-electrode voltage clamp data files were analyzed with custom MATLAB scripts. Scalable vector graphics were produced from MATLAB figures using functions written by Salva Ardid (https://github.com/kupiqu/fig2svg). Rat brain anatomy graphics were derived from "Brain Maps 4.0" (Larry Swanson; University of Southern California; Swanson, 2018).

Hypersensitive b2 nAChR expression
Because most neuronal nAChRs have similar agonist sensitivities, no agonists have been reported that can be delivered to a behaving animal to induce strong (i.e., full agonism) and specific (i.e., no other subtypes affected) activation of b 2 nAChRs. Based on prior work, substitution of a serine at the 99 leucine residue in the second transmembrane a-helix is expected to increase receptor sensitivity sufficiently to allow low concentrations of agonists to be used for selective activation of only those receptors harboring the Leu99 mutation (Labarca et al., 1995(Labarca et al., , 2001Fonck et al., 2003Fonck et al., , 2009Lester et al., 2003;Orb et al., 2004;Tapper et al., 2004;Drenan et al., 2008Drenan et al., , 2010Cohen et al., 2012;Engle et al., 2012Engle et al., , 2013Engle et al., , 2015Wang et al., 2014). To validate the Leu99 mutation in the b 2 subunit, we injected Xenopus oocytes with WT a4 and Leu99Ser b 2 mRNAs and recorded ACh-evoked currents using two-electrode voltage clamp. As expected, oocytes injected with a4b 2Leu99Ser nAChRs (EC 50 : 46 nM; 95% CI: 44-47 nM) were more sensitive to ACh than control a4b 2 nAChRs (EC 50 : 788 nM; 95% CI: 774-802 nM; Fig.  1A). We produced an AAV for expression of b 2Leu99Ser nAChR subunits and a soluble GFP marker. Cre recombinase co-expression is co-injected to flip the b 2-P2A-GFP cassette into the correct orientation relative to the human synapsin promoter (Fig. 1B). We tested this vector by infusing it, together with an AAV driving Cre expression, into the ventral midbrain of adult SD rats (Fig. 1C). This resulted in robust GFP expression in the VTA (Fig. 1D). Although this Leu99Ser validation in Xenopus laevis oocytes. Oocytes were injected with rat nAChR subunit mRNAs: a4 (WT) and b 2 (WT) or a4 (WT) and b 2 (Leu99Ser) in a a:b ratio of 1:2. ACh-evoked inward currents were recorded using two-electrode voltage clamp recordings, and the resulting concentration-response curve is shown for both groups. Representative traces for the indicated ACh concentrations are shown at right for oocytes injected with mRNAs for a4WT b 2WT and a4WT b 2Leu99Ser. B, Design of adeno-associated virus for Cre-dependent expression of (1) b 2Leu99Ser nAChR subunits and (2) GFP. C, AAVs for expression of b 2Leu99Ser subunits (6Cre) were injected bilaterally into the VTA of SD rats. D, Example b 2Leu99Ser GFP reporter expression. After injections described in C, GFP expression was verified in VTA. E, F, Gain-of-function validation in b 2Leu99Ser rats. Brain slices containing the VTA were prepared from naive control rats or b 2Leu99Ser::Cre(1) rats, and patch clamp recordings were made from VTA neurons. ACh (300 mM) approach does not use cell-specific promoters to restrict transgene expression, functional expression of heteromeric nAChRs harboring the b 2Leu99Ser subunit is nonetheless restricted to neurons that also express a4 and/or a6 subunits. These subunits are highly enriched in VTA DA, GABA, and glutamate neurons relative to adjacent brain areas (Azam et al., 2002;Yan et al., 2018). To confirm that this AAV induced a b 2 nAChR gain-of-function, we recorded ACh-evoked inward currents in VTA neurons in brain slices from control and b 2Leu99Ser::Cre(1) rats. At all ACh pulse durations, inward currents in VTA neurons from b 2Leu99Ser::Cre(1) rats were significantly larger than in control rats (100 ms, p = 0.003, one-tailed t test, t =3.386, df=11; 200ms, p = 0.008, onetailed t test, t = 2.848, df = 11; 400 ms, p = 0.006, one-tailed t test, t = 3.026, df = 10; 800 ms, p = 0.009, one-tailed t test, t = 2.901, df = 9; Fig. 1E,F).
b2Leu99Ser rats acquire nicotine SA To determine whether b 2 nAChR activation in the VTA is sufficient for acquisition of nicotine SA, SD rats were infused with b 2Leu99Ser and Cre vectors in the VTA via stereotaxic surgery. Indwelling jugular catheters were placed in a subsequent surgery ( Fig. 2A). Rats were tested for acquisition of nicotine SA using a dose of 1.5 mg/kg/infusion, which is 20-fold lower than the standard training dose (30 mg/kg/inf). After 10 nicotine SA sessions, vehicle (saline) was substituted for nicotine in (n = 7 of 14) of Cre(1) Figure 2. b 2Leu99Ser rats acquire nicotine SA at 1.5 mg/kg/infusion. A, Experimental workflow. Rats were microinjected in VTA with b 2Leu99Ser AAVs, food trained, implanted with jugular catheters, and trained to self-administer nicotine (1.5 mg/kg/infusion; FR1) for 10 d. One subgroup of rats was switched to saline SA for an additional 7 d. A separate FR1 group was transitioned to nicotine SA with a response requirement of FR3 and then FR5. Naive SD rats served as an additional control. B, Mean (6SEM) infusions for nicotine SA at 1.5 mg/kg/inf, followed by saline substitution (in n = 7 of 14 Cre(1) rats). C, Representative 17 d infusion record for a b 2 Leu99Ser rat for 10-d nicotine (1.5 mg/kg/inf) SA and 7-d saline SA. D, Nicotine infusions in b 2Leu99Ser::Cre(1) and Cre(À) rats for acquisition days 6-10. E, Saline substitution in b 2Leu99Ser::Cre(1) rats. Average infusions per animal across days 6-10 before and after the switch to saline in b 2Leu99Ser::Cre(1) rats is shown. Active (F) and inactive (G) responses for infusion data shown in B. H, Mean (6SEM) infusions for nicotine SA at 1.5 mg/kg/inf at response requirements of FR1, FR3, and FR5 for the indicated days. Another group was trained to self-administer saline for 10 d. I, Active and Inactive responses for infusion data shown in H.
Research Article: New Research rats to determine whether nicotine (at 1.5 mg/kg/inf) was reinforcing (Fig. 2B). Control groups included (1) rats infused with b 2Leu99Ser vectors but without Cre vectors (n = 8), and (2) naive SD male rats (n = 9). Whereas these two control groups did not acquire nicotine SA at 1.5 mg/ kg/inf, 14 of 22 b 2Leu99Ser rats with Cre co-expression exhibited acquisition of nicotine SA (Fig. 2B). Eight of 22 b 2Leu99Ser::Cre(1) rats failed to maintain a minimum number of infusions and were considered to have failed to acquire SA. Saline substitution after 10 nicotine SA sessions reduced nicotine self-infusions (Fig. 2B), suggesting that b 2Leu99Ser::Cre(1) rats were responding to 1.5 mg/ kg nicotine because it was reinforcing. A complete infusion record is shown for nicotine SA and saline substitution for a representative b 2Leu99Ser::Cre(1) rat (Fig. 2C). The mean number of nicotine infusions across sessions 6-10 was significantly greater for b 2Leu99Ser rats that received Cre recombinase compared with the Cre(À) group (p = 0.0024, unpaired t test, t = 3.465, df = 20; Fig. 2D). Saline substitution reduced nicotine self-infusions in b 2Leu99Ser:: Cre(1) rats (p = 0.0395, paired t test, t = 2.113, df = 6; Fig.  2E). Active responses largely mimicked the infusion data (Fig. 2F), and none of the three groups had notable responding on the inactive nose poke (Fig. 2G).
To provide additional evidence that b 2Leu99Ser::Cre (1) rats were responding for 1.5 mg/kg nicotine, two additional control experiments were performed. First, nicotine-naive b 2Leu99Ser::Cre(1) rats were trained to selfadminister saline for 10 sessions. Responding for saline declined (Fig. 2H,I), similar to b 2Leu99Ser::Cre(À) and SD control rats self-administering 1.5 mg/kg/inf nicotine, confirming that b 2Leu99Ser::Cre(1) rats were responding for nicotine at 1.5 mg/kg/inf. Second, after 10 SA sessions at FR1, b 2Leu99Ser::Cre(1) rats self-administering 1.5 mg/ kg/inf nicotine were transitioned to a response requirement of FR3 and then FR5. Infusions were maintained after switching rats to FR3 and appeared to decline only slightly, then stabilize, at FR5 (Fig. 2H). Active responses increased during FR3 and FR5 compared with FR1, with no apparent increase in responses on the inactive nose poke (Fig. 2I). These additional controls confirm that b 2Leu99Ser::Cre(1) rats responded specifically for 1.5 mg/ kg/inf nicotine.

DA neuron nAChR activation and nicotine SA
The results from SD rats involve virus injections into VTA, but they do not select for a specific VTA cell type (DA, GABA, glutamate, etc.). Although a majority of VTA neurons are dopaminergic, our results do not rule out the possibility that VTA GABA or other cell types mediate the behavioral response we reported. Next, we tested the hypothesis that activation of b 2* nAChRs on VTA DA neurons is sufficient for nicotine reinforcement. Transgenic Long-Evans rats that express Cre recombinase in tyrosine hydroxylase (TH) neurons (n = 11; Witten et al., 2011) were infused with our Cre-dependent b 2Leu99Ser vector in the VTA via stereotaxic surgery (Fig. 4A), catheterized, and allowed to acquire nicotine SA at a nicotine dose of 1.5 mg/kg/inf (Fig. 4B). Expression in TH-Cre rats resulted in strong GFP reporter expression in neurons that could be co-labeled with anti-TH antibody staining (Fig. 4C). Nontransgenic (non-Tg) littermates of TH-Cre rats (n = 6), infused in the VTA with the same Cre-dependent b 2Leu99Ser vector, served as controls. N = 10 of 11 injected TH-Cre b 2Leu99Ser rats acquired SA (n = 1 of 11 failed acquisition), maintained 15-20 infusions per day through the first 10 d, then increased their mean self-infusions to ;40 infusions by the 17th day (Fig. 4D). By contrast, non-Tg rats microinjected in the VTA with the b 2Leu99Ser vector had very few infusions (,5/d) through day #17 (Fig. 4D). Indeed, TH-Cre rats earned significantly more infusions than non-Tg littermates during days 11-17 (p = 0.0085, unpaired t test, t = 3.062, df = 14; Fig. 4E). To confirm that 1.5 mg/kg/inf nicotine is reinforcing, saline was substituted for nicotine for days 18-24 (Fig. 4D). Compared with their presaline level of self-infusions, TH-Cre b 2Leu99Ser rats significantly reduced their infusions when saline was substituted for nicotine (p = 0.0391, paired t test, t = 2.412, df = 9; Fig. 4F). Active responses largely reflected the infusion data (Fig.  4G), and inactive responses were very low (Fig. 4H).

DA release in b2Leu99Ser rats
Because SA of other psychostimulants (i.e., cocaine) induces changes to dopamine transporter function and evoked dopamine release (Calipari et al., 2013), we asked whether TH-Cre b 2Leu99Ser rats (n = 3) with and without a history of nicotine SA exhibited differential DA dynamics compared with non-Tg rats (n = 3). Following SA, brain slices were prepared containing NAc core (NAcc) and fast scan cyclic voltammetry was used to record electricallyevoked DA release. Peak [DA] achieved by single pulse stimulation (representative traces; Fig. 5A) was significantly reduced in TH-Cre b 2Leu99Ser NAcc slices compared with non-Tg controls (p = 0.007, unpaired t test, t = 2.956, df = 23; Fig. 5B). Nicotine history did not appear to account for this effect in the TH-Cre group, as there was no significant difference in DA release between nicotine-experienced TH-Cre and nicotine-naive TH-Cre rats (Fig. 5B). DA uptake (V max ) was slower in TH-Cre (p = 0.0079, unpaired t test, t = 2.91, df = 23) rats compared with non-Tg controls, which was also not dependent on nicotine history (Fig. 5C). Results in Figure 5A-C were collected with our standard stimulation intensity of 7.5 V Figure 3. Subthreshold nicotine concentrations distinguish b 2Leu99Ser rats after standard nicotine SA. A, Experimental workflow. Rats were microinjected in VTA with b 2Leu99Ser AAVs (6Cre), implanted with jugular catheters, and trained on nicotine SA (30 mg/ kg/inf) for 10 d. Rats were then switched to 1.5 mg/kg/inf nicotine for 7 d. B, Mean (6SEM) infusions nicotine SA at 30 mg/kg/inf, followed by 1.5 mg/kg/inf. C-E, Reduced nicotine concentration enhances nicotine SA in b 2Leu99Ser::Cre(1) rats but reduces SA in controls. Average infusions per animal across days 6-10 before and after the switch to 1.5 mg/kg/inf (days 13-17) in (C) b 2Leu99Ser::Cre(1), (D) b 2Leu99Ser::Cre(À), and (E) SD control rats. F, Nicotine infusions in b 2Leu99Ser::Cre(1) (n = 9) and Cre(À) (n = 6) rats during 1.5 mg/kg/inf after 10 d at 30 mg/kg/inf. G, Predicted CSF nicotine during 30 mg/kg/inf nicotine SA (days 6-10) in b 2Leu99Ser::Cre(1) and Cre(À) rats. Nicotine infusion data from Figure 3B was used to estimate CSF nicotine levels (6SEM, shaded above/below) while rats self-administered 30 mg/kg/inf. H, Predicted CSF nicotine in b 2Leu99Ser::Cre(1) and Cre(À) rats during SA of 1.5 mg/kg/inf. CSF nicotine was determined for days 13-17 as described above (G) for 30 mg/kg/inf. Active (I) and inactive (J) responses for infusion data shown in B. -P2A-GFP was bilaterally microinjected into the VTA of TH-Cre or littermate nontransgenic (non-Tg) rats. B, Experimental workflow. Rats were microinjected in VTA (A), food trained, implanted with jugular catheters, and trained to self-administer nicotine (1.5 mg/kg/inf) for 17 d. Rats were then switched to saline SA for 7 d. C, b 2Leu99Ser-P2A-GFP expression in TH(1) neurons. Four weeks after microinjection (A), VTA sections were co-stained for TH and GFP to confirm GFP expression in TH(1) neurons. Bottom right panels show a single TH neuron expressing b 2Leu99Ser-P2A-GFP. D, Mean (6SEM) infusions nicotine SA (1.5 mg/kg/inf then saline). E, Nicotine infusions (6SEM) in TH-Cre (n = 10) and non-Tg (n = 6) b 2 Leu99Ser rats during 1.5 mg/kg/inf. F, Saline substitution reduces nicotine SA in TH-Cre b 2 Leu99Ser rats. Average infusions across days 11-17 before and after the switch to saline (days 18-24) are shown. Active (G) and inactive (H) responses for infusion data shown in D.
input (350-mA output to the slice). To determine whether reduced DA release in TH-Cre b 2Leu99Ser NAcc was dependent on stimulation intensity, we measured singlepulse evoked DA release at a range of stimulation intensities. Peak DA release in TH-Cre b 2Leu99Ser NAcc was systematically lower for stimulation input intensities from 3-10 V (Fig. 5D), which was not accounted for by nicotine history. Normalized release was similar to controls (Fig. 5E). Next, we determined whether DA release elicited under "phasic-like" stimulation differed in TH-Cre b 2Leu99Ser slices by using a four-pulse stimulation train across varying frequencies. Peak DA release evoked by a four-pulse stimulation train was reduced in TH-Cre b 2Leu99Ser NAcc at 3 Hz (p = 0.038, t = 2.202, df = 23), 30 Hz (p = 0.0197, t = 2.507, df = 23), and 100 Hz (p = 0.0148, t = 2.634, df = 23; Fig. 5F), although normalized DA release in TH-Cre b 2Leu99Ser NAcc was similar to non-Tg controls (Fig. 5G). Phasic-like DA release from TH-Cre b 2Leu99Ser slices was mostly unaffected by nicotine history, though there was a significant difference in normalized release at 10-Hz stimulation between TH-Cre and TH-Cre (nicotine naive; p = 0.0053, unpaired t test, t = 3.093, df = 22; Fig. 5G). Finally, we examined DA release in TH-Cre b 2Leu99Ser NAcc and non-Tg controls following blockade of b 2* nAChRs with dihydro-b -erythroidine (DHb E, 100 nM). DA release evoked by a single stimulation was 51.8 6 2.6% of predrug levels in TH-Cre b 2Leu99Ser NAcc and 45.0 6 3.0% of predrug levels in non-Tg controls (p = 0.1032, t = 1.697, df = 23). DHb E was more effective at reducing DA release following low-frequency (3 and 10 Hz) stimulation trains compared with higher frequency trains (Fig. 5H). Normalized release was very similar for TH-Cre b 2Leu99Ser rats versus non-Tg control rats (Fig. 5I).
Finally, we asked whether DA release was differentially modifiable by nicotine in NAcc slices from b 2Leu99Ser:: Cre(1) SD rats versus naive SD control rats. In control slices, nicotine reduced DA release evoked by single-pulse stimulation and a four-pulse train in a concentration-dependent manner (Fig. 6A,C). DA release from b 2Leu99Ser:: Cre(1) NAcc was more resistant across these nicotine concentrations, resulting in an increased IC 50 for single pulse stimulation and four-pulse train stimulation (Fig. 6B,D).

Discussion
In this study, we demonstrate that expression of a gainof-function b 2 nAChR subunit in the ventral midbrain (VTA) of male rats is sufficient to significantly alter intravenous nicotine SA. Specifically, b 2Leu99Ser VTA expression enables rats to acquire operant responding for nicotine infusions at a dose that is 20-fold lower than the typical training dose. Additional experiments suggest that b 2Leu99Ser VTA expression may shift the dose response curve for nicotine reinforcement to the left. SA experiments in TH-Cre indicate that nAChR activation on VTA DA neurons is sufficient for nicotine reinforcement. b 2Leu99Ser behavioral alterations were accompanied by modifications to electrically evoked DA release. Together, these results contribute novel mechanistic insights into nicotine Figure 5. Altered DA release and uptake in TH-Cre b 2Leu99Ser rats with a history of nicotine SA. After nicotine SA (Fig. 4), NAc core slices were prepared from TH-Cre and non-Tg controls microinjected in VTA with AAV5.2-FLEX-b 2Leu99Ser-P2A-GFP. Another group of AAV5.2-FLEX-b 2Leu99Ser-P2A-GFP-injected TH-Cre animals, but which did not have exposure to nicotine, are included as an additional control. A, Average (6SEM, shaded) DA release trace for the indicated groups. B, C, Electrically evoked (single pulse stimulation) DA release (B) and DA uptake (C). D, E, Input/output relationship for evoked DA release in TH-Cre and non-Tg rats. DA release from NAc core slices was measured at a range of stimulation intensities (0.5-10 V, single pulse), and is expressed as peak [DA] release (D) and normalized DA release I. F, G, DA release after four-pulse stimulation at 3, 10, 30, and 100 Hz is shown as peak [DA] (F) and expressed as a percentage of DA release evoked by single pulse stimulation (G). H, I, Dhb E (100 nM) was bath-applied to the NAc core slices described above, and multipulse stimulation was conducted and reported as described for F, G. reinforcement mechanisms and cholinergic modulation of DA transmission.
Nicotine reinforcement: VTA b2 nAChRs are sufficient Loss-of-function studies using b 2 nAChR antagonists (Corrigall et al., 1994) or b 2 knock-out mice (Maskos et al., 2005;Pons et al., 2008;Tolu et al., 2013) have linked b 2-containing nAChRs with nicotine reinforcement, while others (le Novère et al., 1999;King et al., 2004;Walters et al., 2006;Drenan et al., 2008Drenan et al., , 2010Naudé et al., 2016) have linked nicotine-related phenotypes (place preference, locomotor activation, motivation, etc.) with VTA b 2containing nAChRs. Notably, two prior studies served as a key starting point on which our study builds. Changeux and colleagues reported that re-expression of b 2 subunits in the VTA of b 2 KO mice was sufficient to restore intracranial (intra-VTA) self-administration of nicotine (Maskos et al., 2005). Tolu and colleagues later reported that this restoration of intracranial nicotine self-injection required re-expression of b 2 in both DA and GABA neurons in VTA (Tolu et al., 2013). Our viral-mediated approach in adult rats overcomes several limitations of these important papers, including (1) examination of intravenous nicotine self-administration with saline substitution control experiments capable of detecting nicotine reinforcement, (2) examination of a species (rats) with nicotine metabolism that more closely approaches human nicotine metabolism (Matta et al., 2007), and (3) examination of experimental animals without preexisting global b 2 KO that may cause developmental alterations to reward and/or learning-related circuits. For example, and related to the latter point, global b 2 KO mice have altered passive avoidance learning (Picciotto et al., 1995;King et al., 2003). This study, by examining the effect of selective VTA b 2 nAChR activation, provides a complementary approach to loss-of-function experiments.
Early work in rats established 30mg/kg/inf as an optimal training dose for acquisition of nicotine SA (Corrigall and Coen, 1989;Donny et al., 1995). The dose of nicotine (1.5 mg/kg/inf) that supported acquisition of SA in rats expressing b 2Leu99Ser nAChRs is markedly lower than doses that normally support SA. This sensitization to low nicotine concentrations is further apparent when considering that CSF nicotine levels in rats self-administering 1.5 mg/kg/inf are predicted to be only ;5 ng/ml after a 2-h SA session. CSF nicotine in untargeted control rats or b 2Leu99Ser-expressing rats is predicted to stabilize at 30-50 ng/ml during a 30mg/kg/inf SA session, which is very similar to levels achieved in humans after cigarette smoking (Isaac and Rand, 1972). In 30 mg/kg/inf SA sessions, the predicted CSF versus time profile clearly suggests loading and maintenance behavior (Allain et al., 2015); rats robustly self-administer nicotine within the first 10-20 min, then slow their rate of nicotine self-injections to maintain predicted CSF levels between 30 and 50 ng/ml. b 2Leu99Ser-expressing rats selfadministering 1.5 mg/kg/inf show a somewhat different profile, with a less-pronounced loading phase and stronger boosting of predicted CSF nicotine levels later in the 2-h SA session.
VTA neurons send projections to a variety of brain areas, including NAc, amygdala, prefrontal cortex, hippocampus, and lateral habenula (Lammel et al., 2008;Stamatakis et al., 2013). These neurons include DAergic, GABAergic, glutamatergic, and dual-transmitter cells (Yamaguchi et al., 2007(Yamaguchi et al., , 2015Lammel et al., 2014). b 2-containing nAChRs are expressed both in somatodendritic and presynaptic compartments (Drenan et al., 2008(Drenan et al., , 2010, and systemic nicotine from IVSA likely acts on these presynaptic and/or postsynaptic b 2Leu99Ser nAChR populations to promote reinforcement. Chief among these, based on our results in TH-Cre rats, is the mesolimbic DA pathway that includes, though may not be limited to, VTA to NAc circuits. We noted that, on average, TH-Cre rats expressing b 2Leu99Ser in DA neurons show steady nicotine SA until day ;10 when they escalate their intake. SD rats expressing b 2Leu99Ser nAChRs in all VTA neuron types appear to show a briefer latency to this escalatory behavior. There could be several explanations for this, such as: (1) expression in SD rats may have allowed a greater fraction of VTA neurons to be infected, resulting in more rapid acquisition of nicotine SA; (2) b 2Leu99Ser expression in DA and non-DA (GABA, glutamate, etc.) neurons in SD rats may have invoked additional circuitry that promotes acquisition of nicotine SA. Consistent with the latter, Maskos and colleagues reported that re-expression of b 2 subunits only in VTA DA or GABA neurons in the VTA of b 2 nAChR KO mice is insufficient to restore intra-VTA nicotine self-infusion behavior (Tolu et al., 2013). This suggested that the concerted action of b 2 nAChRs on DA and GABA neurons are necessary for nicotine reinforcement.

nAChR pore mutations and desensitization
Before being expressed in vivo, nAChR subunits with mutations in pore-lining residues (especially the 99 and 139 positions) were thoroughly studied in vitro with a variety of ectopic expression techniques. Cys-loop receptor subunits with 99 and 139 mutations exhibited increases in receptor sensitivity, elimination of inward rectification, and/or changes to single channel properties. These include increased probability of opening and stabilization of the open state (Revah et al., 1991;Bertrand et al., 1992;Yakel et al., 1993;Filatov and White, 1995;Labarca et al., 1995). These and other studies highlighted a key change in nAChR biophysics when the Leu99 residue is modified: slowing or elimination of desensitization. Accordingly, nAChRs incorporating b 2Leu99Ser subunits likely have reduced desensitization when nicotine or ACh is bound. Instead of the normal shutoff of inward current flow in response to desensitization, a large fraction of the b 2Leu99Ser nAChR receptor pool likely retains activity while nicotine is available. Two results in this report, which should be further studied, are consistent with this: (1) inward current responses in VTA neurons from rats expressing b 2Leu99Ser subunits appeared to take longer to return to baseline after a pulse of ACh is delivered (Fig. 1F), and (2) greater concentrations of nicotine are required to attenuate (presumably via receptor desensitization) electrically-evoked DA release in NAcc slices from b 2Leu99Ser rats (Fig. 6). Whereas nicotine concentrations that are normally reinforcing have a rewarding and aversive component because of the balance of activation/desensitization, nicotine's aversive property may be substantially reduced in rats expressing b 2Leu99Ser nAChR subunits in the VTA. Our approach, which can be conceptualized as a knock-down of VTA b 2* nAChR desensitization, suggest that nicotinemediated desensitization of b 2-containing receptors plays an important role in the behavioral pharmacology of nicotine reward and reinforcement. By attenuating desensitization of mesolimbic b 2* nAChRs, nicotine is transformed into a much more potent psychostimulant.

DA release and b2Leu99Ser nAChRs
In TH-Cre rats expressing b 2Leu99Ser nAChRs in VTA, the decrease in evoked DA release amplitude and the reduced DA uptake speed resembles previous results that examined DA release after cocaine SA. Calipari and colleagues reported that five cocaine SA sessions (6-h session, FR1, 1.5 mg/kg/inf, 40 infusion max) were sufficient to significantly reduce single pulse evoked DA release amplitude and velocity of uptake (Calipari et al., 2014). Phillips and colleagues reported that diminished DA signaling is correlated with escalation of cocaine SA, and that boosting DA via L-DOPA reverses escalation (Willuhn et al., 2014). These data suggest that b 2Leu99Ser nAChR subunit expression in VTA neuron somata and/or NAc presynaptic terminals induces neuroadaptations that blunt DA signaling while enhancing nicotine intake behavior. These putative adaptations are at least partially independent of nicotine exposure, as DA release in NAcc of nicotine naive rats expressing b 2Leu99Ser nAChR subunits is similar to release from b 2Leu99Ser rats with a history of nicotine exposure (Fig. 5). Normalized DA release, only at a 10-Hz stimulation frequency, was elevated in nicotine-experienced b 2Leu99Ser rats compared with nicotine-naive rats (Fig. 5G), but this may be because of sampling variation. Many adaptations can explain the blunted DA release phenomenon in b 2Leu99Ser NAcc, such as changes to DA transporter levels/function, changes in presynaptic Ca 21 handling, modulation of vesicular monoamine transporter, modified presynaptic release machinery, altered ACh release from cholinergic interneurons, etc. Future experiments will be required to understand how these adaptations enable rats to acquire and maintain nicotine SA at very low doses of nicotine. Interestingly, relative frequency-dependent increases in DA release are preserved in TH-Cre rats expressing b 2Leu99Ser nAChRs, although absolute DA release is reduced.

Limitations and future studies
Our approach to targeting the mesolimbic pathway in SD rats includes viral transduction of VTA neurons, but we cannot exclude the possibility that nearby circuits also contribute.
DA and/or GABA neurons in the adjacent substantia nigra (pars compacta and pars reticulata) may have been infected, potentially altering nicotine-related behaviors. However, this risk is mitigated by our data from TH-Cre rats; we would not have expected the SA results from TH-Cre rats to corroborate the results from SD rats if b 2Leu99Ser nAChR activation in non-DA neurons plays a role.
Expression of b 2Leu99Ser nAChR subunits in mesolimbic circuitry will provide several opportunities for future mechanistic research. For example, it will enable us to determine whether reinstatement of nicotine seeking occurs after acquisition of nicotine SA purely through the mesolimbic pathway. Moreover, our approach can also be used to determine whether activation of b 2* nAChRs on other circuits is sufficient to support nicotine SA. Rats expressing b 2Leu99Ser nAChR subunits in the VTA could also be used in drug discovery efforts to determine whether lead compounds have abuse liability via activation of b 2* nAChRs.