Neuropsychopharmacology (2010) 35, 665–673 & 2010 Nature Publishing Group All rights reserved 0893-133X/10 $32.00 www.neuropsychopharmacology.org a-Conotoxin MII-Sensitive Nicotinic Acetylcholine Receptors in the Nucleus Accumbens Shell Regulate Progressive Ratio Responding Maintained by Nicotine ,1 1 1 1 Darlene H Brunzell* , Karen E Boschen , Elizabeth S Hendrick , Patrick M Beardsley and 2,3 J Michael McIntosh 1 2 Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, USA; Department of Psychiatry, University of 3 Utah, Salt Lake City, UT, USA; Department of Biology, University of Utah, Salt Lake City, UT, USA b2 subunit containing nicotinic acetylcholine receptors (b2*nAChRs; asterisk (*) denotes assembly with other subunits) are critical for nicotine self-administration and nicotine-associated dopamine (DA) release that supports nicotine reinforcement. The a6 subunit assembles with b2 on DA neurons where a6b2*nAChRs regulate nicotine-stimulated DA release at neuron terminals. Using local infusion of a-conotoxin MII (a-CTX MII), an antagonist with selectivity for a6b2*nAChRs, the purpose of these experiments was to determine if a6b2*nAChRs in the nucleus accumbens (NAc) shell are required for motivation to self-administer nicotine. Long-Evans rats lever-pressed for 0.03 mg/kg, i.v., nicotine accompanied by light + tone cues (NIC) or for light + tone cues unaccompanied by nicotine (CUEonly). Following extensive training, animals were tested under a progressive ratio (PR) schedule that required an increasing number of lever presses for each nicotine infusion and/or cue delivery. Immediately before each PR session, rats received microinfusions of a-CTX MII (0, 1, 5, or 10 pmol per side) into the NAc shell or the overlying anterior cingulate cortex. a-CTX MII dose dependently decreased break points and number of infusions earned by NIC rats following infusion into the NAc shell but not the anterior cingulate a cortex. Concentrations of -CTX MII that were capable of attenuating nicotine self-administration did not disrupt locomotor activity. There was no effect of infusion on lever pressing in CUEonly animals and NAc infusion a-CTX MII did not affect locomotor activity in an open field. These data suggest that a6b2*nAChRs in the NAc shell regulate motivational aspects of nicotine reinforcement but not nicotine-associated locomotor activation. Neuropsychopharmacology (2010) 35, 665–673; doi:10.1038/npp.2009.171; published online 4 November 2009 Keywords: a6; a3; motivation; reward; locomotor activation; tobacco INTRODUCTION self-administration (Corrigall et al, 1992), suggesting that these DA inputs are critical for nicotine reinforcement. Cigarette smoking is the leading preventable cause of death An accumulation of transgenic and neurochemical data in developed countries and is a growing health problem suggests that nicotine-associated DA release and nicotine worldwide (WHO, 2008). Nicotine, the major psychoactive reinforcement are regulated by nicotinic receptors that ingredient in tobacco, and cigarette-associated cues result contain the b2 nicotinic acetylcholine receptor subunit in mesolimblic dopamine (DA) release from the ventral (b2*nAChR; asterisk (*) denotes assembly with other tegmental area (VTA) and activation of VTA projection subunits) (Corrigall et al, 1994; Picciotto et al, 1998; regions including the anterior cingulate cortex and the Champtiaux et al, 2003; Salminen et al, 2004, 2007; Tapper nucleus accumbens (NAc), brain areas associated with the et al, 2004; Maskos et al, 2005; Drenan et al, 2008; Pons et al, rewarding effects of nicotine in smokers (Stein et al, 1998; 2008), making these receptors an attractive therapeutic Schroeder et al, 2001; Due et al, 2002; Volkow et al, 2007). target for tobacco cessation (Gonzales et al, 2006; Rollema Lesions of DA projections to the NAc attenuate nicotine et al, 2007). b2*nAChRs, however, have a ubiquitous expression pattern and contribute more generally to moti- vation, mood, and cognition (for review, see Levin et al, 2006; Brunzell and Picciotto, 2009). Because b2*nAChRs *Correspondence: Dr DH Brunzell, Department of Pharmacology, Virginia Commonwealth University, 410 N 12th Street, PO Box are also very diverse in their composition, identification 980613, Richmond, VA 23298, USA, Tel: + 1 804 628 7584, of receptor subunits that assemble with b2 but that Fax: + 1 804 828 2117, E-mail: [email protected] have a more selective expression pattern may lead to Received 8 July 2009; revised 5 September 2009; accepted 27 tobacco cessation compounds with less potential for side September 2009 effects. a6b2*nAChRs support motivation for nicotine use DH Brunzell et al 666 The a6 nAChR subunit assembles with b2 but is MATERIALS AND METHODS selectively expressed in catecholaminergic nuclei and on terminals in catecholaminergic projection areas (Le Novere Animals et al, 1996; Klink et al, 2001). b2*nAChRs can be classified Twenty-eight adult, male, Long-Evans rats (Harlan Labora- according to their sensitivity to the cone snail toxin, tories, Dublin, VA), weighing approximately 300 g on a-conotoxin MII (a-CTX MII). Synaptosome studies using arrival, were used for these studies. Rats were individually a combination of knockout technology and nicotinic housed in a temperature- and humidity-controlled environ- antagonism with the a-CTX MII peptide indicate that ment with a 12:12 h light/dark cycle (lights on 0600 hours). a6b2*nAChRs on striatal DA terminals participate in Animals began testing at 320 g; animals were nutritionally nicotine-stimulated DA release (Champtiaux et al, 2003; maintained at this body weight by daily food rations Salminen et al, 2004, 2007). Both a3b2* and a6b2* nAChRs throughout behavioral testing (Christian et al, 1998). The are highly sensitive to a-CTX MII antagonism (Cartier experimental protocol was approved by the Institutional et al, 1996; Kuryatov et al, 2000; McIntosh et al, 2004), but Animal Care and Use Committee at Virginia Common- expression of the a6 subunit greatly predominates in the wealth University, and all animals were treated according to VTA and NAc, where the a3 subunit is essentially absent the Guidelines for the Care and Use of Laboratory Animals, (Champtiaux et al, 2002; Whiteaker et al, 2002; Salminen as set forth by the National Institutes of Health. et al, 2005). A recent report by Drenan et al (2008) showed that a single-point mutation in the pore-forming region of Drug Dosing and Administration the a6 subunit, which renders a6b2*nAChRs hypersensitive to nicotine, led to nicotine-associated stimulation of During self-administration procedures, rats received VTA DA neurons and locomotor activation at otherwise 0.03 mg/kg, i.v., infusion of nicotine (by weight of freebase) subthreshold levels of nicotine; these data suggest that in 0.0533 ml delivered over 1 s. Nicotine hydrogen tartrate activation of a6b2*nAChRs are sufficient for DA-stimulat- salt was dissolved in 0.9% sterile saline and stored in ing effects of nicotine. In addition, nicotine self-adminis- the dark to prevent degradation. a-CTX MII was synthe- tration is absent in a6 subunit null mutant mice but sized as previously described (Cartier et al, 1996) and lentiviral rescue of a6 subunit mRNA in the VTA region of dissolved in 0.9% sterile saline. Aliquots were stored at À201C until used for experimentation and were kept these animals leads to recovery of nicotine self-administra- 1 tion in an acute, single-day, tail-vein procedure (Pons et al, at 4 C no longer than 1 week after thawing. Animals 2008). Together these studies suggest that a6b2*nAChRs received 0.5–1 ml intra-accumbens shell infusions of 0, 1, 5, on mesolimblic DA neurons regulate DA release and or 10 pmol per side of a-CTX MII. For locomotor studies, nicotine reward; however, the neuroanatomical locus where rats received s.c. injections of 0.175 mg/kg nicotine (by a6b2*nAChRs exert their effects on nicotine reinforcement weight of freebase) at a concentration of 2.0 mg/ml in sterile and locomotor activation is unclear. In addition, although saline. the tail-vein self-administration paradigm suggests that a b 6 2*nAChRs in the VTA and/or VTA projection areas Intra-Jugular Catheter Implantation may be critical for initiation of tobacco use, less is known regarding the role of these receptors in nicotine reinforce- All surgeries were performed using aseptic procedures. ment following repeated nicotine exposure. The current Anesthesia was induced with 3.5% isoflurane gas at 3.5 liter/ studies used a more sustained nicotine self-administration min oxygen and maintained at 2.0% isoflurane and 2.5 liter/ paradigm in rats. Following training under a fixed ratio min of oxygen. Surgical areas were shaved and cleaned with (FR) schedule of reinforcement, animals were switched 7.5% povidone–iodine and 70% reagent alcohol. A tapered to a progressive ratio (PR) schedule of reinforcement in polyurethane catheter (3.5 French; Access Technologies) was which rats were required to emit an increasing number implanted in the right jugular vein above the atrium. of responses for a single i.v. infusion of nicotine accom- Following implantation, the catheter was passed subcuta- panied by light and tone cues (NIC). To determine if neously to the rat’s back and connected to a back-mounted MII-sensitive nAChRs on terminals in the NAc shell and pedestal (Plastics One, Roanoke, VA). To prevent infection, anterior cingulate cortex regulate motivation for nicotine all rats received s.c. injection of 75 000 U penicillin G and and nicotine-associated cues, we used local infusions 0.1 ml intracatheter injection of 0.031 mg/ml ticarcillin/ of a-CTX MII to block a6b2*nAChRs in these DA projection clavulanate in 25% glycerol/heparinized saline (catheter lock). areas immediately before PR testing. Control animals During surgery, rats received 5 mg/kg, i.p., carprofen for lever-pressed for the same cues as nicotine subjects, but preemptive analgesia. Rats also received 64 mg acetamino- the cues were unaccompanied by nicotine (CUEonly).