Molecular and cellular pharmacology

Bupropion-induced inhibition of α7 nicotinic acetylcholine receptorsexpressed in heterologous cells and neurons from dorsal raphe nucleusand hippocampus

Elizabeth Vázquez-Gómez a, Hugo R. Arias b,n, Dominik Feuerbach c,Marcela Miranda-Morales d, Stefan Mihailescu a, Katarzyna M. Targowska-Duda e,Krzysztof Jozwiak e, Jesús García-Colunga d,nn

a Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, Méxicob Department of Medical Education, California Northstate University College of Medicine, 9700W. Taron Dr., Elk Grove, CA 95757, USAc Neuroscience Research, Novartis Institutes for Biomedical Research, Basel, Switzerlandd Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla 3001,Querétaro 76230, Méxicoe Department of Chemistry, Laboratory of Medicinal Chemistry and Neuroengineering, Medical University of Lublin, Lublin, Poland

a r t i c l e i n f o

Article history:Received 27 January 2014Received in revised form2 June 2014Accepted 20 June 2014Available online 9 July 2014

Keywords:α7 nicotinic acetylcholine receptorAntidepressantsBupropionDorsal raphe nucleusHippocampusMolecular docking

Chemical compounds studied in this article:Bupropion (PubChem CID: 62884)

a b s t r a c t

The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptorsexpressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitoryactivity of bupropion was studied on GH3-α7 cells by Ca2þ influx, as well as on neurons from the dorsalraphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by usinga whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinicacetylcholine receptor was determined by [3H]imipramine competition binding assays and moleculardocking. The fast component of acetylcholine- and choline-induced currents from both brain regionswas inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcho-line receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 mMbupropion, a concentration that could be reached in the brain during clinical administration.Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrationsthat coincide with its inhibitory potency (IC50¼54 mM) and binding affinity (Ki¼63 mM) for α7 nicotinicacetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and moleculardocking results support a luminal location for the bupropion binding site(s). This study may help tounderstand the mechanisms of actions of bupropion at neuronal and molecular levels related with itstherapeutic actions on depression and for smoking cessation.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

Major depression and nicotine addiction are social devastatinghealth situations. There is a high degree of comorbidity betweenmood disorders and tobacco dependence (Killen et al., 2003;Mackowick et al., 2012; Tsuang et al., 2012). Furthermore, theprevalence of nicotine dependence in patients with major depres-sion is greater than among individuals who had never experiencedmajor depression or with no psychiatric diagnosis (Glassman et al.,

1990). Persons that present an adverse impact to smoking cessa-tion treatment have increased risk of experiencing mild to severestates of depression (Covey et al., 1998).

Nicotinic acetylcholine receptors are widely distributed in thenervous system and are implicated in a variety of disorders,including depression and drug addiction (Arias, 2009; Changeux,2010; Mineur and Picciotto, 2010; Shytle et al., 2002; Yakel, 2013).Neurons from the dorsal raphe nucleus and interneurons fromthe stratum radiatum of the hippocampal CA1 region express pre-and post-synaptic nicotinic acetylcholine receptors (Alkondon andAlbuquerque, 1993; Garduño et al., 2012). Interestingly, serotoner-gic projections from the dorsal raphe nucleus to the hippocampusand nucleus accumbens (brain regions implicated in cognitivedeficits, depression, and nicotine addiction) are modulated by

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European Journal of Pharmacology

http://dx.doi.org/10.1016/j.ejphar.2014.06.0590014-2999/& 2014 Elsevier B.V. All rights reserved.

n Corresponding author. Tel.: þ916 686 7300; fax: þ916 686 7310.nn Corresponding author. Tel./fax: þ52 442 238 1063.E-mail addresses: hugo.arias@cnsu.edu (H.R. Arias),

garciacolunga@unam.mx (J. García-Colunga).

European Journal of Pharmacology 740 (2014) 103–111

nicotinic acetylcholine receptors (Aznar et al., 2005; Chang et al.,2011; Mineur and Picciotto, 2010).

One common substance, bupropion, is widely used to clinicallytreat both depression and nicotine addiction (reviewed in Arias(2009), Dhillon et al. (2008), Dwoskin et al. (2006), and Paterson(2009)). Bupropion is a dual inhibitor of noradrenaline anddopamine transporters (Stahl et al., 2004), and also inhibits avariety of nicotinic acetylcholine receptor subtypes in a non-competitive fashion (Arias et al., 2009, 2012; García-Colunga etal., 2011; Slemmer et al., 2000; reviewed in Arias (2009)). How-ever, there are just few reports on the direct effect of bupropion onthe electrical responses elicited by activation of nicotinic acetyl-choline receptors in neurons (Alkondon and Albuquerque, 2005;Mansvelder et al., 2007). In this regard, the aim of this work was tostudy the inhibitory potency of bupropion for α7 nicotinic acet-ylcholine receptors expressed in heterologous cells and comparewith the inhibition elicited on choline-induced currents (i.e., α7-containing nicotinic acetylcholine receptors) in neurons from thedorsal raphe nucleus as well as in interneurons from the stratum-radiatum of the hippocampal CA1 region. Finally, the structuralinteraction of bupropion with the α7 nicotinic acetylcholinereceptor ion channel was determined by [3H]imipramine competi-tion binding and molecular docking methods.

2. Materials and methods

2.1. Ca2þ influx measurements in the GH3-α7 cell line

The inhibitory potency of bupropion for α7 nicotinic acetylcho-line receptors was determined by Ca2þ influx experiments usingthe GH3-α7 cell line as previously described (Arias et al., 2010a).Briefly, 5�104 cells per well were seeded 48 h prior to the Ca2þ

influx experiment on black poly-L-lysine 96-well plates (Costar,Corning Inc., NY, USA) and incubated at 37 1C in a humidifiedatmosphere (5% CO2/95% air). The medium was changed 16 hbefore the experiment to 1% fetal bovine serum in HEPES-buffered salt solution (in mM): 130 NaCl, 5.4 KCl, 2 CaCl2,0.8 MgSO4, 0.9 NaH2PO4, 25 glucose, 20 HEPES, and pH 7.4. Onthe day of the experiment, the medium was removed by flickingthe plates and replaced with 100 ml HEPES-buffered salt solution/1% fetal bovine serum containing 2 μM fluo-4 (Molecular Probes,Eugene, OR, USA) in the presence of 2.5 mM probenecid (Sigma,Buchs, Switzerland). The cells were then incubated at 37 1C ina humidified atmosphere (5% CO2/95% air) for 1 h. Plates wereflicked to remove excess of fluo-4, washed twice with HEPES-buffered salt solution/1% fetal bovine serum, and finally refilledwith 100 ml of HEPES-buffered salt solution containing differentconcentrations of bupropion and pre-incubated for 5 min. Plateswere then placed in the cell plate stage of the fluorimetric imagingplate reader (Molecular Devices, Sunnyvale, CA, USA). A baselineconsisted of five measurements of 0.4 s for each record. (7)‐Epibatidine (0.1 mM) was then added from the agonist plate to thecell plate using the 96-tip pipettor simultaneously to fluorescencerecordings for a total of 3 min. In parallel experiments, 0.1 mM(7)-epibatidine was co-injected with different concentrations ofbupropion. The laser excitation and emission wavelengths were488 and 510 nm, at 1 W, with a CCD camera opening of 0.4 s. Theconcentration–response data were curve-fitted by nonlinear leastsquares analysis using the Prism software (GraphPad Software, SanDiego, CA).

2.2. Preparation of brain slices

All procedures were carried out in accordance with theNational Institute of Health Guide for Care and Use of Laboratory

Animals and were approved by the Institutional Animal CareCommittee of the Universidad Nacional Autónoma de México,with an effort to minimize the number of animals used andtheir suffering. The experiments were performed on brain slicesobtained from Wistar rats, 15–20 postnatal days. Animals weredeeply anesthetized with isoflurane and decapitated. Their brainswere quickly removed and placed into ice cold (4 1C) solutioncontaining (in mM): 250 sucrose, 2.5 KCl, 1.2 NaH2PO4, 5 MgCl2,0.5 CaCl2, 26 NaHCO3, 10 glucose, and pH 7.4. Coronal midbrainslices of 350 mm thick containing the dorsal raphe nucleus orhippocampal area were cut with a Vibratome Leica VT 1000 andsubmerged in artificial cerebrospinal fluid containing (in mM): 125NaCl, 2.5 KCl, 1.23 NaH2PO4, 1 MgCl2, 2 CaCl2, 26 NaHCO3, 10glucose, and pH 7.4. The slices were stabilized in this solution forat least 1 h before transferring a single slice to the recordingchamber. All solutions were continuously bubbled with 95% O2

and 5% CO2 at room temperature.

2.3. Whole-cell patch-clamp recordings

Individual slices were transferred into a tissue chamber andsuperfused throughout the experiment with artificial cerebrosp-inal fluid at a rate of 2–3 ml/min. Whole-cell voltage-clamprecordings (Hamill et al., 1981) were performed with a PC-ONEPatch/Whole Cell Clamp (Dagan Corporation, MN, USA), using anacquisition system Digidata 1440A driven with pClamp 10.

Patch-clamp electrodes were made with borosilicate glass (Sut-ter Instrument, CA, USA) having a resistance of 3–7 MΩ when filledwith either of two internal solutions: (in mM): 140 KCl, 5 NaCl,1 MgCl2, 10 HEPES, 10 EGTA (adjusted pH 7.4 with KOH); or 140 Cs-gluconate, 2 MgCl2, 0.5 CaCl2, 10 HEPES, 5 EGTA, 2 MgATP (adjustedpH 7.4 with CsOH). Similar results were obtained by using indis-tinctly each internal solution. Experimental data were stored in a PCusing a Digidata 1440A AD converter (Axon Instruments, Union City,CA, USA), at a sampling rate of 10 kHz. Individual neurons werevisualized using an infrared video-microscopy system (BX51WI,Olympus Instruments, Japan) endowed with an 80� water immer-sion objective. Neurons selected for recording were located closethe midline area of the dorsal raphe nucleus identified as thetranslucent area between the medial longitudinal fasciculus and theaqueduct; or in the stratum radiatum hippocampal CA1 area. Allrecorded cells were maintained at a potential of �70 mV.

Brief puffs (2–5 psi, 500 ms) of 1 mM acetylcholine or 10 mMcholine on dorsal raphe nucleus or hippocampal neurons wereapplied through a fine tip glass micropipette placed about 10 mmfar from the recorded neuron, by using a microinjector (IM 300,Narisihige Comp., Japan). Different substances were added to theexternal solution (artificial cerebrospinal fluid) during all theexperiments: 500 nM atropine to eliminate the muscarinic compo-nent, the glutamate receptor antagonists, 6-cyano-7-nitroquinoxa-line-2,3-dione (10 mM) and DL-2-amino-5-phosphonopentanoicacid (50 mM), the γ-aminobutyric acid (GABA) A receptor antagonistbicuculline (10 mM), and 200 nM tetrodotoxin to avoid arrivalof action potentials. Repeated administrations of acetylcholineor choline were applied at 5-min intervals to allow recovery ofnicotinic acetylcholine receptors from desensitization. The effects ofbupropion were assessed by microinjections of acetylcholine orcholine on the recording neurons before and after bupropion bathapplication.

We used the pClamp 10 software (Molecular Devices, CA, USA)to measure the acetylcholine- and choline-induced currents inthe absence or presence of burpropion, and Origin 9 (MicrocalSoftware Inc., MA, USA) to analyze and graph the results. Dataare presented as mean7S.E.M. Comparison of the mean valueswas performed by the Student's t-test, with po0.05 consideredstatistically significant.

E. Vázquez-Gómez et al. / European Journal of Pharmacology 740 (2014) 103–111104

2.4. Bupropion-induced inhibition of [3H]imipramine binding to α7nicotinic acetylcholine receptors

To localize the bupropion binding site in the α7 nicotinicacetylcholine receptor, [3H]imipramine was used as a probe forthe antidepressant site as it was determined previously in differ-ent nicotinic acetylcholine receptors (Arias et al., 2010a, 2010b).In this regard, the effect of bupropion on [3H]imipramine bindingto α7 nicotinic acetylcholine receptors was studied using SHSY5Y-α7 cell membranes as described previously (Arias et al., 2010a).More specifically, nicotinic acetylcholine receptor membranes(1.5 mg/ml) were suspended in binding saline buffer (in mM: 50Tris–HCl, 120 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and pH 7.4) with 14 nM[3H]imipramine, and preincubated for 30 min at room tempera-ture. Nonspecific binding was determined in the presence of100 mM imipramine. The total volume was divided into aliquots,and increasing concentrations of bupropion were added to eachtube and incubated for 2 h at room temperature. The radioligandbound to nicotinic acetylcholine receptors was then separatedfrom free ligand by a filtration assay using a 48-sample harvestersystemwith GF/BWhatman filters (Brandel Inc., Gaithersburg, MD,USA), previously soaked with 0.5% polyethylenimine for 30 min.The membrane-containing filters were transferred to scintillationvials with 3 ml of Bio-Safe II (Research Product International Corp,Mount Prospect, IL, USA), and the radioactivity was determinedusing a Beckman LS6500 scintillation counter (Beckman Coulter,Inc., Fullerton, CA, USA).

The concentration–response data were fitted by nonlinear leastsquares analysis using the Prism software, and the correspondingligand concentration that inhibits 50% binding (IC50) and the Hillcoefficient (nH) was calculated. The observed IC50 values weresubsequently transformed into inhibition constant (Ki) valuesusing the Cheng–Prusoff relationship (Cheng and Prusoff, 1973)

Ki ¼ IC50=f1þð½½3H�imipramine�=K imipramined Þg; ð1Þ

where [[3H]imipramine] is the initial concentration of [3H]imipra-mine, and Kd

imipramine corresponds to the dissociation constant of[3H]imipramine for the α7 nicotinic acetylcholine receptor (1 μM;Arias et al., 2010a). The calculated Ki and nH values are summar-ized in Table 1.

2.5. Molecular docking of bupropion enantiomers to the α7 nicotinicacetylcholine receptor ion channel

The model of the α7 nicotinic acetylcholine receptor was builtapplying homology modeling methods using the cryo-electronmicroscopy structure of the Torpedo nicotinic acetylcholine recep-tor determined at �4 Å resolution (PDB ID 2BG9) (Unwin, 2005) asa template, as described previously (Arias et al., 2009, 2010a,2010b, 2012). Modeller 9.9 was used to obtain 100 homology

models, and subsequently, their Discrete Optimized Protein Energy(DOPE) profiles (Eswar et al., 2006) were assessed. The best modelwas subjected to model quality assessment, using the web-basedtools of Verify3D (Eisenberg et al., 1997) and ProCheck (Laskowskiet al., 1993).

Bupropion enantiomers, in the protonated and neutral states,were prepared using Spartan 10V.1.1.0 (Wavefunction, Inc., Irvine,CA, USA), and the molecular docking simulations were performedusing the same protocol as reported previously (Arias et al., 2012).

2.6. Materials

[3H]Imipramine (47.5 Ci/mmol) was obtained from PerkinElmerLife Sciences Products, Inc. (Boston, MA, USA) and stored inethanol at �20 1C. (7)-epibatidine hydrochloride and tetrodo-toxin citrate were obtained from Tocris Bioscience (Ellisville, MO,USA). Fetal bovine serum was purchased from Gibco BRL (Paisley,UK). (7)-bupropion hydrochloride [(7)-2-(tert-butylamino)-1-(3-chlorophenyl)propan-1-one], imipramine hydrochloride, acet-ylcholine chloride, atropine, DL-2-amino-5-phosphonopentanoicacid, bicuculline methobromide, choline chloride, 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt hydrate, and methylly-caconitine citrate hydrate were purchased from Sigma-Aldrich(St. Louis, MO, USA). Salts were of analytical grade.

3. Results

3.1. Inhibitory potency of bupropion at α7 nicotinic acetylcholinereceptors

The potency of (7)-epibatidine to activate α7 nicotinic acetyl-choline receptors was first determined by assessing the fluores-cence change in GH3-α7 cells after (7)-epibatidine stimulation(Fig. 1). The observed EC50 value (ligand concentration producing50% Ca2þ influx) for (7)-epibatidine (5274 nM; Table 1) is inthe same concentration range as those previously determinedfor α7 nicotinic acetylcholine receptors (Arias et al., 2010a).Pre-incubation with bupropion subsequently inhibited the (7)-epibatidine-induced nicotinic acetylcholine receptor activation(Fig. 1). Comparing the calculated IC50 with values from previousstudies (Arias et al., 2009, 2012; reviewed in Arias (2009)), it isapparent that bupropion has low selectivity and low potency forthe α7 nicotinic acetylcholine receptor subtype (Table 1). However,the inhibitory potency increased 3.3-fold when the α7 nicotinicacetylcholine receptors were inhibited by bupropion in the pre-sence of the agonist (co-injection protocol). The observed nH valueis close to unity using the pre-incubation protocol, whereasit is higher than unity using the co-injection protocol (Table 1).This indicates that bupropion pretreatment inhibits α7 nicotinic

Table 1Activation potency of (7)-epibatidine as well as inhibitory potency and binding affinity of (7)-bupropion at α7 nicotinic acetylcholine receptors.

Protocol Conformational state EC50a

(nM)IC50

a

(μM)nH

a,b Kic

(μM)nH

b,c

Ca2þ influx: (7)-epibatidine only Activated 5274 – 3.6970.18 – –

Ca2þ influx: 5 min preincubation with bupropion followed by activation with (7)-epibatidine for several seconds

Mix of different states – 179720 1.0870.04 – –

Ca2þ influx: Co-injection of (7)-epibatidine and bupropion for several seconds Mix of activated anddesensitized states

– 5477 1.4870.07 – –

[3H]Imipramine competition binding Mainly resting state – – – 63710 0.7670.09

a These values were obtained from Fig. 1.b The Hill coefficient.c These values were obtained from Fig. 6.

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acetylcholine receptor activation by a non-cooperative mechan-ism, suggesting that bupropion stabilizes the closed state of thereceptor, whereas bupropion inhibits the recently activated recep-tor by a cooperative mechanism. This latter mechanism suggests

the existence of more than one binding site for bupropion and/orthe combination of several inhibitory processes (e.g., open-channel blockade and induced receptor desensitization), as it hasbeen observed for bupropion and its derivatives in muscle nico-tinic acetylcholine receptors (Arias et al., 2009, 2012; reviewed inArias (2009)).

3.2. Dorsal raphe and hippocampal neurons express functional α7and non-α7 nicotinic acetylcholine receptors

To compare the inhibitory activity of bupropion on α7 nicotinicacetylcholine receptors expressed in heterologous cells with thoseendogenously expressed in neurons, we subsequently determinedits pharmacological activity in neurons from the dorsal raphenucleus and in interneurons from the stratum radiatum of thehippocampal CA1 region. The identity of endogenous nicotinicacetylcholine receptors was determined by comparing the elec-trical activity elicited by the unspecific agonist acetylcholine withthat for the selective agonist choline, as well as by the inhibitoryeffect provoked by methyllycaconitine, a selective antagonist forα7 nicotinic acetylcholine receptors (Alkondon et al., 1992, 1997),on these agonist-induced electrical responses.

The local application of 1 mM acetylcholine or 10 mM cholineonto hippocampal and dorsal raphe nucleus neurons generatedan inward current that decayed in the presence of the agonist, dueto receptor desensitization (Fig. 2A). Of 15 recorded dorsal raphenucleus neurons, 11 presented acetylcholine-induced currents,

Fig. 1. Effect of bupropion on (7)-epibatidine-induced Ca2þ influx in GH3-α7cells. Increased concentrations of (7)-epibatidine (■) activate α7 nicotinic acetyl-choline receptors. Subsequently, cells were pre-treated (5 min) with severalconcentrations of bupropion followed by addition of 0.1 μM (7)-epibatidine (○),or alternatively different concentrations of bupropion were co-injected with 0.1 μM(7)-epibatidine (□). The response was normalized to the maximal (7)-epibatidineresponse which was set as 100%. The plots show the results of 27 (■), 11 (○), and 8(□) separate determinations. Error bars correspond to the S.D. The calculated EC50,IC50, and nH values are summarized in Table 1.

Fig. 2. Functional activity of α7-containing nicotinic acetylcholine receptors in dorsal raphe nucleus and hippocampal neurons. (A) Upper traces correspond to acetylcholine(ACh) induced currents recorded in hippocampal (Hipp, left) and dorsal raphe nucleus (DRN, right) neurons. Lower traces correspond to choline (Ch) induced currentsrecorded in hippocampal (left) and DRN (right) neurons. (B) Inhibition of acetylcholine- and choline-induced currents by 5 nM methyllycaconitine (MLA). The upperacetylcholine-induced currents were recorded from a hippocampal CA1 interneuron (n¼5), whereas the lower choline-induced currents from a dorsal raphe nucleus neuron(n¼3). Ion currents on the left correspond to controls, middle currents were obtained in the presence of methyllycaconitine after 7 min of preincubation, and the records onthe right illustrate the recovered responses after 30 min of wash out methyllycaconitine. The holding potential was �70 mV. During all the experiments, slices werecontinuously perfused with the external solution plus (in mM): 10 6-cyano-7-nitroquinoxaline-2,3-dione, 50 DL-2-amino-5-phosphonopentanoic acid, 0.2 tetrodotoxin,0.5 atropine, and 10 bicuculline. The time of agonist application (500 ms, 2–5 psi) is illustrated by a line above the left upper record in (A). Calibration bars: 100 pA applies forthe record having the scale; 50 pA for the rest of the records.

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ranging from 30 to 400 pA; whereas in 42 out of 50 hippocampalneurons tested the acetylcholine-induced currents ranged from 10to 512 pA. On the other hand, choline-induced currents werepresent in 11 out of 26 dorsal raphe nucleus neurons, rangingfrom 20 to 400 pA; and in 31 out of 37 hippocampal interneuronsrecorded, ranging from 20 to 525 pA. The high variability in theamplitude of agonist-induced currents may indicate the presenceof different nicotinic acetylcholine receptor subtypes with variableexpression among neurons. A fast component in acetylcholine-induced currents (Fig. 2A, upper records) and a fast decayinginward current generated by choline (Fig. 2A, bottom records)were observed in dorsal raphe nucleus and hippocampal neurons.Afterward, methyllycaconitine was applied at concentrationsbetween 5 and 20 nM. The choline-induced current was comple-tely inhibited by 20 nM methyllycaconitine (n¼4; data notshown). In addition, 5 nMmethyllycaconitine completely inhibitedthe fast, but not the slow, inward component of the acetylcholine-induced currents (Fig. 2B, upper records), whereas the choline-induced currents were reversibly inhibited (Fig. 2B, lower records).These results indicate that both the fast acetylcholine- and thetotal choline-induced currents were due to the activation of α7-containing nicotinic acetylcholine receptors.

3.3. Bupropion inhibits nicotinic acetylcholine receptors from dorsalraphe nucleus and hippocampal neurons

To evaluate the direct interaction of bupropion with nicotinicacetylcholine receptors, the effects of bupropion were explored onboth acetylcholine- and choline-induced currents in the dorsalraphe nucleus and CA1 hippocampal region from rat acute slices.Focal 1 mM acetylcholine applications (2–5 psi, 500 ms) wereevaluated before, during, and after the acute bath application of50 mM bupropion during 7–8 min. The acetylcholine-induced cur-rents were inhibited during and even after bupropion exposure,and partially recovered after washing out bupropion (Fig. 3).Similar results were obtained on choline-induced currents inthe dorsal raphe nucleus and CA1 hippocampal region (Fig. 4).In a set of experiments, the inhibitory effects of bupropion oncholine-induced currents were compared to those mediated by

methyllycaconitine and α-bungarotoxin, two selective α7 nicotinicacetylcholine receptor antagonists (Alkondon et al., 1992;Couturier et al., 1990). The choline-induced current in hippocam-pal interneurons was inhibited by 50 mM bupropion. The ratiobetween the inhibited choline-induced current by bupropion andthe control current in hippocampal interneurons was 0.5270.04 (n¼6). Then, after recovery of bupropion-inhibited choline-induced current, 5 nM methyllycaconitine (n¼3) or 20 nMα-bungarotoxin (n¼3) for 15 min was added to the bath perfusion.The choline-induced current was completely inhibited by bothantagonists (Fig. 4A and B), confirming the involvement ofα7-containing nicotinic acetylcholine receptors.

To determine whether the observed bupropion-induced inhibi-tion is elicited at clinically relevant concentrations (i.e., �20 mM;reviewed in Arias (2009)), bupropion was tested at this concentra-tion level (10–20 mM). The results show that bupropion effectivelyinhibits choline-induced currents at these concentrations (Fig. 4Cand D). Accordingly, the ratio between the inhibited choline-induced current by 20 mM bupropion and the control current inhippocampal interneurons was 0.6670.06 (n¼3), whereas theratio for the acetylcholine-induced current inhibited by 10 mMbupropion in dorsal raphe nucleus neurons was 0.7270.04 (n¼3).

A comparison of the results between acetylcholine- andcholine-induced currents in the dorsal raphe nucleus and CA1hippocampal region indicates a similar inhibitory activity exertedby 50 mM bupropion (Fig. 5). All data were calculated consideringthe maximal inhibitory effect of bupropion and the maximalrecovery (see Figs. 3 and 4). The height of the columns representsthe amplitude of the agonist-induced currents in the presenceof bupropion normalized to control agonist-induced currents(considered as the unity). The obtained ratios were statisticallydifferent (po0.05): 0.4870.05 and 0.4770.04 for the acetylcholine-induced currents in dorsal raphe nucleus and hippocampal neurons,respectively; and 0.4870.04 for the choline-induced currents inhippocampal interneurons. Very few dorsal raphe nucleus neurons(two from 11) responded to choline with amplitudes high enoughto evaluate confidently the inhibition elicited by bupropion (with aratio of 0.46).

3.4. Binding affinity of bupropion for the α7 nicotinic acetylcholinereceptor ion channel

The binding affinity of bupropion for the α7 nicotinic acetylcho-line receptor ion channel was determined by using [3H]imipramineas a probe for the ion channel as described previously (Arias et al.,2010a). Bupropion inhibits the specific binding of [3H]imipramine tothe α7 nicotinic acetylcholine receptor in a concentration-dependentfashion (Fig. 6). The calculated binding affinity of bupropion for the[3H]imipramine binding site(s) at α7 nicotinic acetylcholine receptorsin the resting state (Ki¼66 μM; Table 1) is relatively lower than thatdetermined at other nicotinic acetylcholine receptor subtypes (Ariaset al., 2009, 2012; reviewed in Arias (2009)). The calculated nH valuefor bupropion is close to unity, indicating that this antidepressantinhibits [3H]imipramine binding to α7 nicotinic acetylcholine recep-tors in a non-cooperative manner, and suggesting that bupropionbinds to the luminal [3H]imipramine site by a steric mechanism. Inturn, this result supports the idea that bupropion interacts within theα7 nicotinic acetylcholine receptor ion channel, as it was previouslyshowed for muscle nicotinic acetylcholine receptors (Arias et al.,2009, 2012; reviewed in Arias (2009)).

3.5. Molecular docking of bupropion enantiomers to the α7 nicotinicacetylcholine receptor

Since bupropion is �95% protonated at physiological pH [pKa¼8.770.4; calculated by the ACD/ADME Suite software (Advanced

Fig. 3. Bupropion decreases acetylcholine-induced currents in dorsal raphe nucleusneurons. (A) Representative acetylcholine (ACh) induced currents from eightseparate experiments, recorded at 4-min intervals. (B) The amplitude of acetylcho-line-induced currents as a function of time, before, during, and after the applicationof bupropion. Ion currents were elicited by a 5-psi, 500-ms puff of 1 mMacetylcholine. The timing of bupropion application is indicated by the thickblack lines.

E. Vázquez-Gómez et al. / European Journal of Pharmacology 740 (2014) 103–111 107

Chemistry Development, Inc., Toronto, Canada)], the moleculardocking is shown for the protonated state. The docking resultsindicate that both bupropion enantiomers interact with two sites:

a site close to the extracellular mouth of the receptor, and anotherin the middle of the channel lumen (Fig. 7). In the former site, (S)-(þ)-bupropion interacts predominantly by van der Waals contacts

Fig. 4. Bupropion decreases choline-induced currents in hippocampal CA1 interneurons. (A, C) Representative choline (Ch) induced currents recorded at 5-min intervalsfrom six and three separate experiments, respectively. (B, D) The amplitude of choline-induced currents is plotted as a function of time, before, during, and after theapplication of bupropion. (A, B) α-bungarotoxin was added to bath perfusion after recovery of bupropion-inhibited choline-induced currents (n¼3). Ion currents wereelicited by a 5-psi, 500-ms puff of 10 mM choline. The timing of bupropion and α-bungarotoxin application is indicated by the thick black lines.

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with residues at position 200 (i.e., Glu259) (outer ring), 170 (i.e.,Leu256) (nonpolar ring), 140 (i.e., Phe253), and 130 (i.e., Val252)(valine ring). In the latter site, (S)-(þ)-bupropion interacts by vander Waals contacts with Phe253, Val252, and Leu248 at theleucine ring (position 90) (Fig. 7B). In addition, the ligand isstabilized by three hydrogen bonds, two formed between itsamino moiety and the hydroxyl groups of two Ser249 (position100) and a third one formed between its oxygen and the samehydroxyl groups. The (R)-(�)-enantiomer only interacts with thesite located in the middle of the ion channel (data not shown).

4. Discussion

Since we do not have a clear picture of how bupropionproduces its pharmacological activity at neuronal level, the inhi-bitory activity of this antidepressant was compared between

heterologous cells and hippocampal interneurons and dorsalraphe nucleus neurons. Additional radioligand binding and mole-cular docking experiments were performed to determine itsbinding site location.

Our Ca2þ influx results (Table 1), and the fact that thebupropion-inhibited choline-induced current in hippocampalinterneurons and dorsal raphe nucleus neurons is sensitive tothe selective antagonists methyllycaconitine and α-bungarotoxinindicate that bupropion inhibits, at least partially, α7 nicotinicacetylcholine receptors. These results are in agreement with previ-ous studies (Slemmer et al., 2000; reviewed in Arias (2009)),indicating that, although with low potency, bupropion inhibits α7nicotinic acetylcholine receptors at clinically relevant concentra-tions (10–20 μM) (Fig. 4C and D). This evidence coincides with theattained brain concentration of bupropion (�20 mM; reviewed inArias (2009)). Interestingly, the fact that ketamine produces rapidand sustained antidepressant effects in patients with severeand treatment-resistant depression (Cornwell et al., 2012), andthat its metabolites strongly inhibit α7 nicotinic acetylcholinereceptors (Moaddel et al., 2013), supports the possibility that α7nicotinic acetylcholine receptors are target for structurally differ-ent antidepressants.

The docking results suggest that both bupropion enantiomersbind to the α7 nicotinic acetylcholine receptor channel, morespecifically between the valine (position 130) and the leucine(position 90) rings (Fig. 7), concurrent with that observed in othernicotinic acetylcholine receptor subtypes (Arias et al., 2009, 2012).Interestingly, the bupropion site overlaps that for imipramine(Arias et al., 2009, 2010a, 2010b, 2012), which is in agreementwith the radioligand competition results, indicating that bupro-pion binds to the [3H]imipramine binding site at the α7 nicotinicacetylcholine receptor (Table 1).

Some of the dorsal raphe nucleus neurons, containing pre- andpost-synaptic functional α4β2 and α7 nicotinic acetylcholinereceptors, project to the nucleus accumbens and hippocampus,impacting serotonin release (Aznar et al., 2005; Chang et al., 2011).Furthermore, during nicotine withdrawal, a persistent serotoner-gic tone is observed increasing anxiety (Cheeta et al., 2001). Thisserotonin increase, in turn, augments dopaminergic transmission(Benloucif et al., 1993). In this regard, bupropion may increasedopamine levels through inhibition of nicotinic acetylcholinereceptors in dorsal raphe nucleus neurons by modulating seroto-nin release. All these neurochemical actions could explain, at leastpartially, the clinical use of bupropion for depression and smokingcessation.

We also found that bupropion inhibits α7-containing nicotinicacetylcholine receptors expressed in GABAergic interneuronsfrom the CA1 hippocampus area. The activation of α7 nicotinicacetylcholine receptors expressed in interneurons can produceinhibition or disinhibition of pyramidal GABAergic neurons (Ji andDani, 2000; Banerjee et al., 2012). Consequently, the observedbupropion-induced inhibition might indirectly stimulate (i.e., dis-inhibit) pyramidal neurons. In this regard, we speculate that thismechanism can be part of the antidepressant activity elicited bybupropion. The results that reduced GABA levels in plasma andcerebrospinal fluid are pronounced in treatment-resistant depres-sion patients (Luscher et al., 2011), and that reduced GABAergicactivity can induce diverse mood disorders (Earnheart et al., 2007)may support our conjecture.

We conclude that bupropion, at concentrations that could bereached in the brain during clinical administration, inhibitsagonist-induced currents mediated by α7 nicotinic acetylcholinereceptors in dorsal raphe nucleus neurons as well as in interneur-ons from the stratum radiatum of the hippocampal CA1 area,and may modulate serotonin signaling and GABA synaptic trans-mission, respectively. This study may help to understand the

Fig. 5. Bupropion inhibits acetylcholine- and choline-induced currents in dorsalraphe nucleus (DRN) and hippocampal (Hipp) neurons. The height of the columns(IExp/ICtrl) represents the minimal amplitude of acetylcholine (ACh) or choline (Ch)induced currents by the action of bupropion normalized to those in the absence ofbupropion considered as the unity (IBP/ICtrl, light gray columns); and the maximalrecovery of the acetylcholine- or choline-induced currents after washing outbupropion compared to those in the absence of bupropion (IRec/ICtrl, dark graycolumns). Data are the mean7S.E.M. The numeral in parenthesis indicates thenumber of experiments; the Student's t-test, npo0.05.

Fig. 6. Bupropion-induced inhibition of [3H]imipramine binding to α7 nicotinicacetylcholine receptors. Nicotinic acetylcholine receptor membranes (1.5 mg/ml)were equilibrated (2 h) with 14 nM [3H]imipramine, and increasing concentrationsof bupropion. Nonspecific binding was determined at 100 mM imipramine. The plotis the combination of two separate experiments, each one in triplicate, where theerror bars correspond to the S.D. The IC50 and nH values were obtained by nonlinearleast-squares fit and the Ki value was calculated using Eq. (1) and summarized inTable 1.

E. Vázquez-Gómez et al. / European Journal of Pharmacology 740 (2014) 103–111 109

mechanisms of actions of bupropion at neuronal and molecularlevels related with its therapeutic actions on depression and forsmoking cessation.

Acknowledgments

This work was supported by a Grant from Dirección General deAsuntos del Personal Académico (DGAPA), UNAM, IN201313 (to J.G.C.),and by the TEAM research subsidy from the Foundation for PolishScience (TEAM 2009/4-5) (to K.J.). E.V.G. was a postdoctoral fellow

from DGAPA, UNAM. We are grateful to Martín García Servín for hisassistance in taking care of the rats.

References

Alkondon, M., Albuquerque, E.X., 1993. Diversity of nicotinic acetylcholine receptorsin rat hippocampal neurons. I. Pharmacological and functional evidence fordistinct structural subtypes. J. Pharmacol. Exp. Ther. 265, 14551473.

Alkondon, M., Albuquerque, E.X., 2005. Nicotinic receptor subtypes in rat hippo-campal slices are differentially sensitive to desensitization and early in vivofunctional up-regulation by nicotine and to block by bupropion. J. Pharmacol.Exp. Ther. 313, 740–750, http://dx.doi.org/10.1124/jpet.104.081232.

Fig. 7. Molecular docking of protonated (S)-(þ)-bupropion within the α7 nicotinic acetylcholine receptor ion channel. (A) Side view of the two binding modes of (S)-(þ)-bupropion within the ion channel: one closer to the extracellular mouth (gray) and another located in the middle of the ion channel (black). Residues from each ring areshown explicitly in stick mode. (B) Detailed view of the two binding sites. (S)-(þ)-bupropion interacts with a site located closer to the extracellular mouth, predominantly byvan der Waals contacts with residues at position 200 (i.e., Glu259) (outer ring), 170 (i.e., Leu256) (nonpolar ring), 140 (i.e., Phe253), and 130 (i.e., Val252) (valine ring). (S)-(þ)-bupropion also interacts with a site located in the middle of the ion channel, by van der Waals contacts with Phe253, Val252, and Leu248 at the leucine (position 90) ring.In addition, the ligand is stabilized by three hydrogen bonds (see black arrows), two formed between its amino moiety and the hydroxyl groups of two Ser249 (position 100),and a third one formed between its oxygen and the same hydroxyl groups. All nonpolar hydrogens atoms are hidden. The ligands are rendered in ball (A) or stick (B) mode.Subunits are shown in secondary structure mode. One subunit was removed for clarity. All other segments (A) or the M2 transmembrane helices (B) are shown in light gray.

E. Vázquez-Gómez et al. / European Journal of Pharmacology 740 (2014) 103–111110

Alkondon, M., Pereira, E.F, Cortes, W.S., Maelicke, A., Albuquerque, E.X., 1997.Choline is a selective agonist of α7 nicotinic acetylcholine receptors in the ratbrain neurons. Eur. J. Neurosci. 12, 2734–2742, http://dx.doi.org/10.1111/j.1460-9568.1997.tb01702.x.

Alkondon, M., Pereira, E.F., Wonnacott, S., Albuquerque, E.X., 1992. Blockade ofnicotinic currents in hippocampal neurons defines methyllycaconitine as apotent and specific receptor antagonist. Mol. Pharmacol. 41, 802–808.

Arias, H.R., 2009. Is the inhibition of nicotinic acetylcholine receptors by bupropioninvolved in its clinical actions? Int. J. Biochem. Cell Biol. 41, 2098–2108, http://dx.doi.org/10.1016/j.biocel.2009.05.015.

Arias, H.R., Feuerbach, D., Targowska-Duda, K.M., Aggarwal, S., Lapinsky, D.J.,Jozwiak, K., 2012. Structural and functional interaction of (7)-2-(N-tert-butylamino)-30-iodo-40-azidopropiophenone, a photoreactive bupropion deri-vative, with nicotinic acetylcholine receptors. Neurochem. Int. 61, 1433–1441,http://dx.doi.org/10.1016/j.neuint.2012.10.011.

Arias, H.R., Feuerbach, D., Targowska-Duda, K.M., Russell, M., Jozwiak, K., 2010a.Interaction of selective serotonin reuptake inhibitors with neuronal nicotinicacetylcholine receptors. Biochemistry 49, 5734–5742, http://dx.doi.org/10.1021/bi100536t.

Arias, H.R., Rosenberg, A., Targowska-Duda, K.M., Feuerbach, D., Jozwiak, K.,Moaddel, R., Wainer, I.W., 2010b. Tricyclic antidepressants and mecamylaminebind to different sites in the human α4β2 nicotinic receptor ion channel. Int.J. Biochem. Cell Biol. 42, 1007–1018, http://dx.doi.org/10.1016/j.biocel.2010.03.002.

Arias, H.R., Gumilar, F., Rosenberg, A., Targowska-Duda, K.M., Feuerbach, D.,Jozwiak, K., Moaddel, R., Wainer, I.W., Bouzat, C., 2009. Interaction of bupropionwith muscle-type nicotinic acetylcholine receptors in different conformationalstates. Biochemistry 48, 4506–4518, http://dx.doi.org/10.1021/bi802206k.

Aznar, S., Kostova, V., Christiansen, S.H., Knudsen, G.M., 2005. α7 nicotinic receptorsubunit is present on serotonin neurons projecting to hippocampus andseptum. Synapse 55, 196–200, http://dx.doi.org/10.1002/syn.20108.

Banerjee, J., Alkondon, M., Pereira, E.F., Albuquerque, E.X., 2012. Regulation ofGABAergic inputs to CA1 pyramidal neurons by nicotinic receptors andkynurenic acid. J. Pharmacol. Exp. Ther. 341, 500–509, http://dx.doi.org/10.1124/jpet.111.189860.

Benloucif, S., Keegan, M.J., Galloway, M.P., 1993. Serotonin-facilitated dopaminerelease in vivo: pharmacological characterization. J. Pharmacol. Exp. Ther. 265,373–377.

Chang, B., Daniele, C.A., Gallagher, K., Madonia, M., Mitchum, R.D., Barrett, L.,Vezina, P., McGehee, D.S., 2011. Nicotinic excitation of serotonergic projectionsfrom dorsal raphe to the nucleus accumbens. J. Neurophysiol. 106, 801–808,http://dx.doi.org/10.1152/jn.00575.2010.

Changeux, J.P., 2010. Nicotine addiction and nicotinic receptors: lessons fromgenetically modified mice. Nat. Rev. Neurosci. 11, 389–401, http://dx.doi.org/10.1038/nrn2849.

Cheeta, S., Irvine, E.E., Kenny, P.J., File, S.E., 2001. The dorsal raphé nucleus isa crucial structure mediating nicotine's anxiolytic effects and the developmentof tolerance and withdrawal responses. Psychopharmacology (Berl) 155, 78–85,http://dx.doi.org/10.1007/s002130100681.

Cheng, Y., Prusoff, W.H., 1973. Relationship between the inhibition constant (Ki) andthe concentration of inhibitor which causes 50 per cent inhibition (IC50) of anenzymatic reaction. Biochem. Pharmacol. 22, 3099–3108.

Cornwell, B.R., Salvadore, G., Furey, M., Marquardt, C.A., Brutsche, N.E., Grillon, C.,Zarate Jr., C.A., 2012. Synaptic potentiation is critical for rapid antidepressantresponse to ketamine in treatment-resistant major depression. Biol. Psychiatry72, 555–561, http://dx.doi.org/10.1016/j.biopsych.2012.03.029.

Couturier, S., Bertrand, D., Matter, J.M., Hernandez, M.C., Bertrand, S., Millar, N.,Valera, S., Barkas, T., Ballivet, M., 1990. A neuronal nicotinic acetylcholinereceptor subunit (α7) is developmentally regulated and forms a homo-oligomeric channel blocked by α-BTX. Neuron 5, 847–856.

Covey, L.S., Glassman, A.H., Stetner, F., 1998. Cigarette smoking and major depres-sion. J. Addict. Dis. 17, 35–46, http://dx.doi.org/10.1300/J069v17n01_04.

Dhillon, S., Yang, L.P., Curran, M.P., 2008. Bupropion: a review of its use in themanagement of major depressive disorder. Drugs 68, 653–689.

Dwoskin, L.P., Rauhut, A.S., King-Pospisil, K.A., Bardo, M.T., 2006. Review of thepharmacology and clinical profile of bupropion, an antidepressant and tobaccouse cessation agent. CNS Drug Rev. 12, 178–207.

Earnheart, J.C., Schweizer, C., Crestani, F., Iwasato, T., Itohara, S., Mohler, H., Lüscher, B.,2007. GABAergic control of adult hippocampal neurogenesis in relation tobehavior indicative of trait anxiety and depression states. J. Neurosci. 27,3845–3854, http://dx.doi.org/10.1523/JNEUROSCI.3609-06.2007.

Eisenberg, D., Lüthy, R., Bowie, J.U., 1997. VERIFY3D: assessment of protein modelswith three-dimensional profiles. Methods Enzymol. 277, 396–404, http://dx.doi.org/10.1016/S0076-6879(97)77022-8.

Eswar, N., Webb, B., Marti-Renom, M.A., Madhusudhan, M.S., Eramian, D., Shen, M.-Y., Pieper, U., Sali, A., 2006. Comparative protein structure modeling usingModeller. Curr. Protoc. Bioinform. 15, http://dx.doi.org/10.1002/0471250953.bi0506s15 (5.6.1–5.6.30).

García-Colunga, J., Godoy-García, U., Vázquez-Gómez, E., 2011. Interactionof bupropion and zinc with neuronal nicotinic acetylcholine receptors. Neuro-pharmacology 61, 1202–1209, http://dx.doi.org/10.1016/j.neuropharm.2011.07.009.

Garduño, J., Galindo-Charles, L., Jiménez-Rodríguez, J., Galarraga, E., Tapia, D.,Mihailescu, S., Hernandez-Lopez, S., 2012. Presynaptic α4β2 nicotinic acetyl-choline receptors increase glutamate release and serotonin neuron excitabilityin the dorsal raphe nucleus. J. Neurosci. 32, 15148–15157, http://dx.doi.org/10.1523/JNEUROSCI.0941-12.2012.

Glassman, A.H., Helzer, J.E., Covey, L.S., Cottler, L.B., Stetner, F., Tipp, J.E., Johnson, J.,1990. Smoking, smoking cessation, and major depression. J. Am. Med. Assoc.264, 1546–1549, http://dx.doi.org/10.1001/jama.1990.03450120058029.

Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-freemembrane patches. Pflugers Arch. 391, 85–100, http://dx.doi.org/10.1007/BF00656997.

Ji, D., Dani, J.A., 2000. Inhibition and disinhibition of pyramidal neurons byactivation of nicotinic receptors on hippocampal interneurons. J. Neurophysiol.83, 2682–2690.

Killen, J.D., Fortmann, S.P., Schatzberg, A., Hayward, C., Varady, A., 2003. Onset ofmajor depression during treatment for nicotine dependence. Addict. Behav., 28,461–470, http://dx.doi.org/10.1016/S0306-4603(01)00266-0.

Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M., 1993. PROCHECK: aprogram to check the stereochemical quality of protein structures. J. Appl.Crystallogr. 26, 283–291.

Luscher, B., Shen, Q., Sahir, N., 2011. The GABAergic deficit hypothesis of majordepressive disorder. Mol. Psychiatry 16, 383–406, http://dx.doi.org/10.1038/mp.2010.120.

Mackowick, K.M., Lynch, M.J., Weinberger, A.H., George, T.P., 2012. Treatment oftobacco dependence in people with mental health and addictive disorders. Curr.Psychiatry Rep. 14, 478–485, http://dx.doi.org/10.1007/s11920-012-0299-2.

Mansvelder, H.D., Fagen, Z.M., Chang, B., Mitchum, R., McGehee, D.S., 2007.Bupropion inhibits the cellular effects of nicotine in the ventral tegmentalarea. Biochem. Pharmacol. 74, 1283–1291, http://dx.doi.org/10.1016/j.bcp.2007.07.034.

Mineur, Y.S., Picciotto, M.R., 2010. Nicotine receptors and depression: revisiting andrevising the cholinergic hypothesis. Trends Pharmacol. Sci. 31, 580–586, http://dx.doi.org/10.1016/j.tips.2010.09.004.

Moaddel, R., Abdrakhmanova, G., Kozak, J., Jozwiak, K., Toll, L., Jimenez, L.,Rosenberg, A., Tran, T., Xiao, Y., Zarate, C.A., Wainer, IW., 2013. Sub-anestheticconcentrations of (R,S)-ketamine metabolites inhibit acetylcholine-evokedcurrents in α7 nicotinic acetylcholine receptors. Eur. J. Pharmacol. 698, 228–-234, http://dx.doi.org/10.1016/j.ejphar.2012.11.023.

Paterson, N.E., 2009. Behavioural and pharmacological mechanisms of bupropion'santi-smoking effects: recent preclinical and clinical insights. Eur. J. Pharmacol.603, 1–11, http://dx.doi.org/10.1016/j.ejphar.2008.12.009.

Shytle, R.D., Silver, A.A., Lukas, R.J., Newman, M.B., Sheehan, D.V., Sanberg, P.R.,2002. Nicotinic acetylcholine receptors as targets for antidepressants. Mol.Psychiatry 7, 525–535, http://dx.doi.org/10.1038/sj.mp.4001035.

Slemmer, J.E., Martin, B.R., Damaj, M.I., 2000. Bupropion is a nicotinic antagonist.J. Pharmacol. Exp. Ther. 295, 321–327.

Stahl, S.M., Pradko, J.F., Haight, B.R., Modell, J.G., Rockett, C.B., Learned-Coughlin, S.,2004. A review of the neuropharmacology of bupropion, a dual norepinephrineand dopamine reuptake inhibitor. Prim. Care Companion J. Clin. Psychiatry 6,159–166.

Tsuang, M.T., Francis, T., Minor, K., Thomas, A., Stone, W.S., 2012. Genetics ofsmoking and depression. Hum. Genet. 131, 905–915, http://dx.doi.org/10.1007/s00439-012-1170-6.

Unwin, N., 2005. Refined structure of the nicotinic acetylcholine receptor at 4 Åresolution. J. Mol. Biol. 346, 967–989, http://dx.doi.org/10.1016/j.jmb.2004.12.031.

Yakel, J.L., 2013. Cholinergic receptors: functional role of nicotinic ACh receptors inbrain circuits and disease. Pflugers Arch. 465, 441–450, http://dx.doi.org/10.1007/s00424-012-1200-1.

E. Vázquez-Gómez et al. / European Journal of Pharmacology 740 (2014) 103–111 111