The α3β4* nicotinic acetylcholine receptor subtype mediates nicotine reward and physical nicotine withdrawal signs independently of the α5 subunit in the mouse
Highlights
► AuIB, a selective α3β4 antagonist, was used to assess α3β4 in nicotine behaviors. ► α3β4 nicotinic receptors do not mediate acute nicotine behaviors. ► α3β4 nicotinic receptors mediate nicotine reward and withdrawal independent of α5. ► α3β4 nicotinic receptors are involved in somatic, but not affective withdrawal.
Introduction
A major in nicotine research is to gain a better understanding of the molecular and receptor mediated mechanisms of nicotine dependence and withdrawal in order to generate more effective smoking cessation therapies. The initial targets for nicotine are nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels which have been identified throughout the central and peripheral nervous systems, as well as at skeletal neuromuscular junctions. While animal studies implicate a significant role for β2-containing nAChRs in nicotine reward, dependence, and withdrawal (Picciotto et al., 1998; Walters et al., 2006; Jackson et al., 2008, 2009b), to date, the 15q25 gene cluster, which contains the CHRNA5/CHRNA3/CHRNB4 genes, coding for the α5, α3, and β4 nAChR subunits respectively, has shown the most robust findings in human genetic studies as a candidate region contributing to risk of heavy smoking, nicotine dependence, and smoking related diseases in humans (Berrettini et al., 2008; Bierut et al., 2008; Liu et al., 2010; Lips et al., 2010; Ware et al., 2011).
The α5 nAChR subunit co-assembles with α3β4* nAChR subtypes (where * denotes the possible inclusion of additional subunits) to form functional receptors in the peripheral ganglia, as well as centrally, in the medial habenula (MHb) and interpeduncular nucleus (IPN) (Wada et al., 1990; Zoli et al., 1995; Quick et al., 1999; Whiteaker et al., 2002). Despite findings of robust associations with the 15q25 gene cluster, rodent studies assessing the role of α3, β4, and α5 nAChRs are limited, though available studies do implicate a role for these nAChRs in nicotine mediated behaviors.
In the central nervous system (CNS), the mesolimbic dopamine pathway contains some α3β4* (where * denotes the possible inclusion of additional subunits) subtypes; however, the majority of this receptor subtype resides in the medial habenula (MHb) and interpeduncular nucleus (IPN) (Wada et al., 1990; Zoli et al., 1995; Quick et al., 1999; Whiteaker et al., 2002). In these brain regions, the α5 nAChR subunit co-assembles with α3β4* nAChR subtypes to various degrees (15–35% of the α3β4* subtypes at the MHb and IPN, respectively) (Grady et al., 2009). Importantly, α5-containing nAChRs in the MHb and IPN have recently been implicated in nicotine self-administration and reward. α5 nAChR knockout (−/−) mice are less sensitive to the acute behavioral effects of nicotine, and maintain expression of a significant nicotine conditioned place preference (CPP) at nicotine doses that are aversive in wild-type (+/+) littermates (Jackson et al., 2010). An increased nicotine intake was also observed in α5 nAChR −/− mice. This effect was ‘rescued’ in −/− mice by re-expressing α5 subunits in the MHb, and recapitulated in rats through α5 subunit knockdown in the MHb (Fowler et al., 2011). In addition, mice null for the α5 nAChR subunit exhibit a reduction in nicotine withdrawal somatic signs (Jackson et al., 2008; Salas et al., 2009).
The role of α3 and β4 nAChR subunits in nicotine's behavioral effects is starting to emerge. Studies using nicotinic knock-out mice, α3 and β4 nAChRs were found to be necessary for nicotine-induced seizures in mice (Kedmi et al., 2004; Salas et al., 2004a, Salas et al., 2004b), while β4 nAChRs partially mediate acute nicotine-induced hypolocomotion (Salas et al., 2004a, Salas et al., 2004b), hypothermic response (Sack et al., 2005), and antinociception (Semenova et al., 2012). Furthermore, withdrawal somatic signs and hyperalgesia are reduced in β4 nAChR −/− mice after nicotine withdrawal (Salas et al., 2004a, Salas et al., 2004b; Stoker et al., 2012). β4 nAChR subunits were also found to be involved in affective nicotine withdrawal measures, as β4 nAChR −/− mice do not display elevated intracranial self-stimulation thresholds following nicotine withdrawal (Stoker et al., 2012). Currently, evidence of a clear behavioral role for α3 nAChR subunits in nicotine withdrawal is lacking. Selective α3β4* antagonists are becoming available to delineate the role of α3β4* nAChR subtypes in nicotine dependence. For example, the peptide α-conotoxin AuIB (AuIB), which was originally isolated from the venom of the predatory cone snail, Conus aulicus, was shown to most potently block α3β4 nAChRs as expressed in Xenopus oocytes (Luo et al., 1998). The peptide was subsequently synthesized, and synthetic material was used in subsequent studies to block native CNS α3β4 nAChRs in hippocampus (Luo et al., 1998; Kulak et al., 2001), locus coeruleus (Fu et al., 1999), interpeduncular nucleus (Grady et al., 2001), and medial habenula (McCallum et al., 2012). More recently, AT-1001, a relatively selective α3β4* antagonist, was reported to dose-dependently block nicotine intravenous self-administration in rats after systemic injection (Toll et al., 2012).
Because of the apparent role of the α3β4* nAChR subtypes in nicotine dependence, the goal of the current study was to better evaluate the involvement of this subtype in nicotine mediated behavioral responses. Thus, we used the α3β4* selective antagonist AuIB to question α3β4* nAChR contributions in acute nicotine behaviors, nicotine reward using the CPP model, and physical (somatic signs, hyperalgesia) and affective (anxiety-related behavior) nicotine withdrawal using a spontaneous nicotine withdrawal model. As an additional measure of affective nicotine withdrawal, we assessed the effects of AuIB in the conditioned place aversion (CPA) model. Further, we determined the contributions of α5 subunits to the effects of the selective α3β4* antagonist AuIB to nicotine reward and somatic nicotine withdrawal by measuring expression of nicotine CPP and nicotine withdrawal somatic signs in α5 nAChR −/− mice after pre-treatment with AuIB.
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Animals
Male adult C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice null for the α5 nicotinic receptor subunit (C57BL/6 background) and +/+ littermates were shipped from Baylor College of Medicine, Houston, Texas (see Salas et al., 2003 for information regarding initial breeders) and were subsequently bred in an animal care facility at Virginia Commonwealth University. Mutant and +/+ mice were obtained by breeding heterozygote pairs. Male adult α5 nAChR +/+ and −/− mice
AuIB has no significant effect on acute nicotine-mediated behaviors
C57Bl/6J mice were treated with AuIB (14 or 70 pmol, i.c.v.) 5 min prior to a single injection of nicotine and tested in a battery of behavioral tests. Results are shown in Table 1. Nicotine produced significant antinociception indicated by a decrease in latency in the tail-flick (F(5,30) = 36.45, p < 0.0001) and hot plate (F(5,30) = 95.00, p < 0.0001) tests, a significant decrease in body temperature (F(5,30) = 291.51, p < 0.0001), and a decrease in locomotor activity (F(5,30) = 17.45, p
Discussion
The goal of the present study was to elucidate the role of α3β4* nAChRs in nicotine mediated behaviors. These behavioral studies using pharmacological agents and genetically modified mice suggest that α3β4* nAChRs are not involved in acute nicotine behaviors, but mediate nicotine reward and physical, but not affective nicotine withdrawal signs. Interestingly, the α5 nAChR subunit, which can co-assemble with the α3β4* subtype to form functional receptors, is not required in this receptor
Acknowledgments
This research was supported by grant # DA-12610 to MID.
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