Nicotine Administration in the Cholinergic Basal ForebrainIncreases Alcohol Consumption in C57BL/6JMice

Rishi Sharma, Pradeep Sahota, and Mahesh M. Thakkar

Background: Alcohol and nicotine are the most commonly abused drugs. The frequent co-morbid-ity of alcohol and nicotine addiction has led to the hypothesis that they may act via a common sub-strate: the nicotinic acetylcholine receptors (nAChRs) especially a4b2 and a7 subtypes, the mostprevalent nAChRs in the brain. Compelling evidence suggests that alcohol enhances the function ofa4b2 subtype. The FDA approved smoking cessation drug, varenicline (“Chantix”), a partial agonist ofa4b2 nAChR subtype, reduces alcohol self-administration and alcohol craving in humans and rodents.The cholinergic basal forebrain (BF) controls various functions including arousal, attention, and cogni-tion, and there is a predominance of a4b2 and a7 subtypes. We have shown that the BF has an impor-tant role in mediating the effects of alcohol and local infusion of nicotine in the BF activates nucleusaccumbens. Does BF have any role in mediating the effect of nicotine on alcohol consumption? Thisstudy was designed to address this question.

Methods: Under standard surgical procedure, C57BL/6J mice were stereotaxically implanted withbilateral stainless steel guide cannula above the BF. Following post operative recovery and habituation,the animals were exposed to the “drinking-in-the-dark” paradigm whereby alcohol (20%) was pre-sented for 2 hours daily for 3 days. On the fourth day, nicotine or artificial cerebrospinal fluid (ACSF)was microinjected bilaterally in the BF. After 1 hour, mice were exposed to alcohol and allowed to self-administer for 4 hours. The effect of BF nicotine infusion on sucrose consumption was also examined.On completion, mice were euthanized, brain removed and processed to localize the BF injection sites.

Results: As compared with the ACSF, bilateral nicotine injections into the BF significantly(p < 0.05; n = 5/group) increased alcohol consumption. Sucrose consumption remained unaffected.

Conclusions: Based on our results, we believe that the BF may have an important role in nicotine–alcohol co-use.

Key Words: Nicotine, Basal Forebrain, Microinjection, Mice, Alcohol.

ALCOHOL AND NICOTINE, an addictive substancein tobacco smoking, are among the most common

co-abused drugs. Approximately, 90% of alcoholics smoke,whereas approximately 60% of smokers binge drink or con-sume substantial amount of alcohol (Batel et al., 1995;DiFranza and Guerrera, 1990). Early onset of smoking inadolescents predicts the development of alcohol-relatedproblems in later life (Grant, 1998; Riala et al., 2004). Theamount of alcohol consumed and the degree of alcoholdependence is positively correlated with the amount oftobacco consumed (Barrett et al., 2006). In contrast, smok-ing cessation reduces alcohol consumption (Fucito et al.,2011; McKee et al., 2009; Mitchell et al., 2012). While

nicotine is believed to be one of the greatest risk factors forthe development of alcoholism, little is known about under-lying neurobiological mechanisms responsible for nicotineand alcohol co-abuse (Davis and de Fiebre, 2006).

It has been suggested that nicotine and alcohol may act viaa common neurological pathway: the nicotinic acetylcholinereceptors (nAChRs; Davis and de Fiebre, 2006). ThenAChRs are ligand-gated ion channels that are activated bythe neurotransmitter, acetylcholine and nicotine. The nAC-hRs in the central nervous system are pentameric combina-tions, formed from a portfolio of genetically distinct a- andb-subunits (a2 to a10 and b2 to b4). Differential associationof various subunits confers distinct functional and structuralproperties to the resultant nAChRs subtype. The most abun-dant nAChR subtypes in the brain are the heteromeric a4b2and homomeric a7 (Millar and Gotti, 2009).

Human genetic association studies implicateCHRNA5–CHRNA3–CHRNB4 gene cluster, encoding forthe a3, a5, and b4 nAChRs subunits, in nicotine and alcoholdependency (Hong et al., 2010; Joslyn et al., 2008; Wanget al., 2009). However, compelling clinical and preclinicalevidence suggest that alcohol enhances the function of a4b2subtype, inhibits a7 subtype and has minimal effects on a3b2subtype (Blomqvist et al., 1992; Butt et al., 2003, 2004;Cardoso et al., 1999; Davis and de Fiebre, 2006; de Fiebre

From the Department of Neurology (RS, PS, MMT), Harry S. Tru-man Memorial Veterans Hospital, University of Missouri, Columbia,Missouri.

Received for publication November 4, 2013; accepted December 21,2013.

Reprint requests: Mahesh M. Thakkar, PhD, Harry S. TrumanMemorial Veterans Hospital Research, Room A023, 800 Hospital Drive,Columbia, MO 65201; Tel.: 573-882-3135; Fax: 573-884-4249; E-mail:’thakkarmm@health.missouri.edu

Published 2014. This article is a U.S. Government work and is in thepublic domain in the U.S.A.

DOI: 10.1111/acer.12353

Alcohol Clin Exp Res,Vol 38, No 5, 2014: pp 1315–1320 1315

ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH Vol. 38, No. 5May 2014

and de Fiebre, 2005; Narahashi et al., 2001; Secko, 2005;Taslim and Dar, 2010; Taslim et al., 2008). Furthermore,varenicline, a partial agonist of a4b2 containing nAChRsreduces alcohol-seeking and consumption in rodents(Chatterjee and Bartlett, 2010; Steensland et al., 2007). Thus,while there is compelling evidence implicating nAChRs inalcohol and nicotine co-abuse, it is yet unclear as to where inthe brain nicotine acts to affect alcohol consumption.

Recently we have demonstrated that the cholinergic basalforebrain (BF) has an important role in mediating thebehavioral effects of alcohol (Sharma et al., 2010a,b, 2012;Thakkar et al., 2010). Moreover, the BF neurons exhibithigh expressions of a4b2 and a7 nACHRs subtypes (Azamet al., 2003) and systemic administration of nicotinic recep-tor agonist activates BF neurons (Biton et al., 2007; Thom-sen et al., 2010). The BF has strong connections withseveral brain regions implicated in alcohol and nicotineaddiction. For example, the BF receives strong dopaminer-gic projections from the ventral tegmental area (VTA) andGABAergic projections from the nucleus accumbens and isthe major source of cholinergic inputs to several brainregions including the cortex and amygdala (Zaborszkyet al., 2012). Furthermore, bilateral BF infusion of nicotinecoupled with systemic alcohol activates nucleus accumbens(Dumontier et al., 2012).

Does nicotine act via the BF to promote alcohol consump-tion? To address this issue, we examined the effects of localadministration of nicotine into the BF on alcohol consump-tion in C57BL/6J mice.

MATERIALS ANDMETHODS

Animals

Adult male C57BL/6J mice (7 to 8 weeks old; 22 to 26 g; JacksonLaboratories, Bar Harbor, ME) were housed under ambient roomtemperature (25 � 2°C), reverse 12 to 12 hour light/dark cycle(light onset = 10:00 PM) and with ad libitum food and water for1 week before experiments were begun. All experimental procedureswere approved by the Institutional Animal Care and Use Commit-tee of Harry S. TrumanMemorial Veterans Hospital.

Drugs

Alcohol solution (20%, v/v) was prepared from ethanol (EtOH;200 proof; Fisher Scientific, Pittsburgh, PA) in tap water. Sucrose(D-sucrose; Fisher Scientific) solution (10%, w/v) was prepared intap water. Stock solution (1 mM) of (�)-Nicotine hydrogen tartrate(Sigma-Aldrich Co. LLC, St. Louis, MO) was prepared with artifi-cial cerebrospinal fluid (ACSF; 147 mM NaCl, 3 mM KCl,1.2 mM CaCl2 and 1.0 mM MgCl2; pH 7.0), aliquoted and storedat �20°C. All working solutions were prepared fresh on the day ofthe experiment.

Surgery

Under isoflurane anesthesia and sterile conditions, mice (n = 20)were stereotaxically implanted with bilateral stainless steel guidecannula (27 G), 2.0 mm above the target site (target coordinates:Anterior = 0.0 mm; Lateral = �1.5 mm and Ventral = 5.3 mm,relative to the bregma and skull surface; Franklin and Paxinos,

2008). Two anchor screws were also implanted, and the entireassembly was fixed onto the skull with dental cement. A 31-gaugestainless steel stylet was inserted to maintain the patency.Subcutaneous flunixin (2.5 mg/kg/12 h for 1 day) was used as apostoperative analgesic. The animals were observed until ambula-tory and then each mouse was singly housed in the experimentalcage (similar to normal mouse cage with 1 grommeted hole on theshorter side for dispensing water/alcohol) and allowed to recoverfor 5 to 7 days.

Alcohol Consumption

We used the drinking-in-the-dark (DID) procedure as describedpreviously (Rhodes et al., 2005, 2007). In brief, 2.5 hours after darkonset, water bottles were removed from animal cages. A single pre-weighed 15 ml plastic bottle with metal sipper tubes containing20% alcohol was introduced into each mouse cage after 30 minutes.Mice were allowed to consume alcohol for 2 hours after which thealcohol bottles were replaced with original water bottles. Subse-quently, alcohol bottles and animals were weighed to calculate theamount of alcohol consumed (g/kg of the body weight). The sameprocedure was repeated on days 2, 3, and 4, except on day 4, micehad access to 20% alcohol for 4 hours.

Drug Infusions

To reduce stress, all mice were habituated to the infusion proce-dures by performing sham-injections on days 2 and 3. The sham-injection protocol was identical to the microinjection protocol andperformed at the same time (1 hour before alcohol exposure)except that the sham-injector was shorter (remained 1.5 mm abovethe target site; to avoid damaging target sites) and no fluid wasinfused.

On day 4, 1 hour before the onset of alcohol exposure, mice wererandomly divided into 2 groups. The ACSF group (n = 5) was bilat-erally infused with ACSF (100 nl/side). The Nicotine group (n = 5)was bilaterally infused with nicotine (10 pmol [equivalent to 1.6 ngof nicotine-free base]/100 nl/side) into the BF. The 10 pmol dose ofnicotine was selected because this dose has been shown to producemaximal effects when infused locally in select brain region (Smithand Dar, 2007; Taslim et al., 2011). The infusion protocol was asdescribed previously (Thakkar et al., 2010). In brief, mouse wasremoved out of its cage and gently held to remove the stylus andinsert the injector cannula (connected to a 0.5 ll Hamilton syringe[Hamilton, Reno, NV] via FEP connector [Eicom, San Diego, CA])into the ipsilateral guide cannula. Once the injector was in place,ACSF or nicotine was slowly microinjected. On completion, theinjector cannula was left in place for an additional 1 minute beforeretracting. The same protocol was repeated to infuse ACSF or nico-tine on the contralateral side. On completion, the animal wasreturned back to its cage. The entire infusion protocol was com-pleted in approximately 5 minutes.

Blood Alcohol Concentration

Blood alcohol concentration (BAC) was measured as describedpreviously (Sharma et al., in press). In brief, on day 4, immediatelyafter the measurement of alcohol consumption, mice were removedfrom their cages, and a small amount (25 ll) of blood was removedfrom the tail vein and centrifuged to separate plasma. The plasmawas used for BAC analysis by using EtOH measurement kit as permanufacturer’s instructions (Genzyme, Framingham,MA).

Sucrose Consumption

To validate the specificity of the effect on alcohol consumption,we examined the effects of bilateral BF nicotine infusion on sucrose

1316 SHARMA ET AL.

(10%) consumption (DID protocol) in separate groups (ACSF andNicotine; 5/group) of 10 mice.

Localization of Injection Sites

On completion of the experiments, mice were euthanizedby sequential transcardiac perfusion with 40 ml of 0.9% coldsaline and 10% formalin (Fisher Scientific). The brains wereremoved, postfixed, equilibrated (20% sucrose in 0.1 Mphosphate buffered saline; pH 7.4) and serially sectioned(40 lm; 3 series) with freezing microtome. One serieswas used for choline acetyl transferase immunohistochemistryto localize the injection site in the BF (Sharma et al., 2010a).

Statistics

Unpaired t-test (Prism; Graphpad Software, La Jolla, CA)was used to examine the effects of BF nicotine infusion onalcohol/sucrose consumption (a = 0.05). Pearson’s correlationwas used to examine the dependence of BAC on alcoholconsumption.

RESULTS

Localization of Injection Sites

All injections sites (n = 20) were localized in the BF region(between AP = 0.26 and �0.10) and are described in 1coronal schematic (AP = 0.14) in Fig. 1A (adapted fromfigure 30; see Franklin and Paxinos, 2008). A representativephotomicrograph depicting the bilateral injection sites inthe midst of cholinergic neurons in the BF is shown inFig. 1B.

Fig. 1. Localization of injections sites. (A) Schematic representation ofall injections sites (n = 20) mapped on 1 coronal schematic (AP = 0.14;adapted from figure 30; see Franklin and Paxinos, 2008) is described. Allinjection sites were localized in the cholinergic basal forebrain (BF;between AP = 0.26 and �0.10). Circles denote alcohol consumption(Black circle = ACSF infusion; Red circle = nicotine infusion); Starsdenote sucrose consumption (Black star = ACSF infusion; Red star =nicotine infusion). (B) A representative photomicrograph depicting thebilateral lesions caused due to microinjections in the BF (large arrowhead)in the midst of ChAT “+ve” cholinergic neurons. Abbreviations: aca,anterior commissure, anterior part; VP, Ventral Pallidum; 3V, thirdventricle. Calibration bar = 100 lm. ACSF, artificial cerebrospinal fluid;ChAT, choline acetyl transferase.

Fig. 2. Effect of basal forebrain (BF) nicotine infusion on alcohol con-sumption. (A) Bilateral microinjections of nicotine (10 pmol) into the BF sig-nificantly increased alcohol consumption. (B) The blood alcoholconcentration (BAC) paralleled alcohol consumption. Mice infused with nic-otine in the BF displayed significantly higher BAC as compared to miceinfused with ACSF. (C) Nicotine (10 pmol) infusion in the BF had no effecton sucrose consumption. ACSF, artificial cerebrospinal fluid.

NICOTINE INCREASES ALCOHOL CONSUMPTION 1317

Alcohol Consumption

As compared to the ACSF infusions (3.2 � 0.4 g/kg),bilateral BF nicotine infusion significantly (t = 2.6, df = 8,p < 0.05; unpaired t-test) increased the amount of alcoholconsumed (4.5 � 0.3 g/kg) during 4 hours of alcohol accesson day 4 (Fig. 2A).

Blood Alcohol Concentration

The BAC levels were significantly (Pearson r = 0.95;p < 0.0001; n = 10) correlated with the amount of alcoholconsumption on day 4. As compared to bilateral BF infusionof ACSF (73.4 � 5.7 mg/dl) BAC was significantly (t = 2.8,df = 8, p < 0.05; unpaired t-test) higher in mice infused withnicotine (105 � 9.8 mg/dl; Fig. 2C).

Sucrose Consumption

In contrast to alcohol concentration, bilateral BF nicotineinfusions did not alter sucrose consumption. Mice infusedwith ACSF consumed 119 � 13.6 ml/kg and mice infusedwith nicotine consumed 110 � 4.3 ml/kg during 4 hours ofsucrose exposure on day 4 (Fig. 2B).

DISCUSSION

The results of our study suggest that nicotine infusion intothe cholinergic BF increases alcohol consumption implicat-ing the role of BF in alcohol and nicotine co-abuse. Infusionof nicotine in the BF had no effect on sucrose consumptionfurther validating the specificity of the effect on alcoholconsumption.

In the present study, we have used “drinking-in-the-dark” paradigm to examine the alcohol consumptionfollowing BF nicotine infusion. Originally designed toexamine binge-like alcohol consumption, the DID modelutilizes the predisposition of C57BL/6J mice to voluntarilyconsume large quantities of alcohol within 2 to 4 hourstime, resulting in a pharmacologically relevant BAC(Rhodes et al., 2005, 2007). Importantly, no prior trainingis required to habituate the animals to high concentrationof alcohol (Sprow and Thiele, 2012). The DID paradigmhas been extensively used to examine the central effects ofseveral neurotransmitters/neuropeptides on alcohol con-sumption (Linsenbardt and Boehm, 2009; Lowery et al.,2010; Lowery-Gionta et al., 2012; Moore and Boehm,2009; Sparrow et al., 2012; Sparta et al., 2013). Ample evi-dences suggest that the dopaminergic VTA has a criticalrole in mediating the effects of nicotine and alcohol. Forexample, infusion of nicotine into the VTA enhances alco-hol-induced increase in accumbal dopamine (Tizabi et al.,2002). This effect is blocked by infusion of nicotinic recep-tors antagonists in the VTA (Ericson et al., 2003). Fur-thermore, the VTA of alcohol preferring P rats showsenhanced sensitivity to the reinforcing effects of nicotine

suggesting a genetic linkage between high alcohol prefer-ence and the sensitivity of the VTA toward reinforcingactions of nicotine (Hauser et al., 2013). Finally, Roguskiand colleagues (2013) have recently demonstrated thatconcurrent gestational exposure to alcohol and nicotinedisrupts the dopaminergic circuitry in the VTA resultingin increased nicotine self-administration in adolescents.

Although the mechanism of how nicotine infusion in theBF increases alcohol consumptions is unclear and underinvestigation, it is interesting to note that the BF has a pre-dominance of a4b2 and a7 subtypes of nAChRs, and boththese receptors play a pivotal role in mediating alcohol con-sumption (Davis and de Fiebre, 2006). Varenicline, a partialagonist of a4b2 containing nAChRs reduces alcohol con-sumption in humans and rodents (Chatterjee and Bartlett,2010; Fucito et al., 2011; McKee et al., 2009; Mitchell et al.,2012; Steensland et al., 2007). Systemic injection of cytisine,a partial agonist of a4b2 containing nAChRs nicotinic recep-tor also reduces alcohol consumption (Hendrickson et al.,2009). Local BF infusion of nicotine coupled with systemicalcohol administration activates nucleus accumbens, anessential brain region in the mesolimbic dopaminergic path-way associated with rewarding and reinforcing effects ofboth alcohol and nicotine (Dumontier et al., 2012; Ericsonet al., 2003; Tizabi et al., 2002).

Ongoing studies in our laboratory are investigating theeffects of selective nicotinic antagonists such as methyllyca-conitine (selective antagonist for a7 containing nAChRs)and dihydro b-erythroidine (a selective antagonist for a4b2containing nAChRs) on alcohol consumption to examine thebasal and/or tonic involvement of BF nicotinic receptors innicotine and alcohol co-abuse.

In conclusion, the results of our study suggest that the nic-otine infusion into the cholinergic BF increases alcohol con-sumption suggesting that the BF may have a role in nicotineand alcohol co-abuse.

ACKNOWLEDGMENTS

We thank Shafi Lodhi, Kevin Bradshaw, and DavidDeRoode for helping us with microinjection and immunohis-tochemistry, and Carrie Harris for animal care. This workwas supported by the Harry S. Truman Memorial VeteransHospital, and funding from National Institute of AlcoholAbuse and Alcoholism (AA020334 & AA0174720).

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