Previously, the nonspecific nicotinic receptor antagonist, mecamylamine, has been shown to reduce ethanol intake in rats that have learned to drink ethanol through at least two week training with increasing concentration of free or limited access ethanol (Blomqvist et al. 1996
; Le et al. 2000
)). In addition, mecamylamine has been reported to reduce the subjective euphoria of ethanol in humans (Blomqvist et al. 1996
; Chi and de Wit 2003
; Le et al. 2000
). To our knowledge, this is the first report that nAChR blockade reduces ethanol consumption in mice during the DID paradigm, a model of binge drinking where C57BL/6J mice consume alcohol until intoxicated. Mecamylamine dose-dependently reduced alcohol intake and this also lead to a significant reduction in blood-ethanol concentration suggesting that mecamylamine was not inhibiting the metabolism of ethanol. Sucrose intake, however, was not reduced indicating specificity for alcohol consumption and not a general effect on reward signaling. Reduction of ethanol intake by mecamylamine was mediated by blockade of neuronal nAChRs expressed in the CNS because the non-specific nAChR antagonist, hexamethonium, did not significantly alter alcohol consumption. Prior studies indicate that mecamylamine delivered systemically or directly into the VTA blocks elevation of ethanol-mediated dopamine release in the nucleus accumbens (Blomqvist et al. 1993
; Blomqvist et al. 1997
). Thus, it is likely that mecamylamine is reducing ethanol intake via a similar mechanism in the DID assay. Although there have been reports that high doses of mecamylamine can non-competitively inhibit NMDA receptors (Fu et al. 2008
; O’Dell and Christensen 1988
), we observe a decrease in the volume of ethanol consumption at doses as low as 0.5 mg/kg suggesting that mecamylamine is acting via blockade of neuronal nAChRs..
Because of the vast array of nAChR subtypes expressed in the CNS, identifying the specific composition of receptors involved in ethanol reinforcement is a difficult, but important question. High affinity α4β2 and low affinity α7 nAChRs are two of the most abundant nicotinic receptors in the CNS and could represent potential candidates for at least partially mediating ethanol reward, α4β2 in particular since these receptors have been clearly implicated in nicotine dependence (Picciotto et al. 1998
; Tapper et al. 2004
). However, the α4β2 selective and α7 selective antagonists dhβe and MLA, respectively, both of which readily cross the blood-brain barrier, failed to significantly reduce ethanol intake. These data support prior studies that have shown little effect of these compounds on both operant responding, ethanol-mediated dopamine release in nucleus accumbens, and ethanol self-administration in rats (Le et al. 2000
; Soderpalm et al. 2000
). The straightforward interpretation of these data would be that α4β2 and α7 nAChRs are not involved in alcohol self-administration. However, caution in this interpretation is warranted especially in regard to higher affinity heteromeric nicotinic receptors that could contain α4β2 in addition to a third or even fourth subunit that may render them relatively insensitive to dhβe (Salminen et al. 2004
Interestingly, acute exposure to nicotine dose dependently reduced alcohol intake in the DID paradigm. This is in opposition to at least one previous study that indicates that nicotine can enhance ethanol intake in rats in a restricted access drinking assay (Smith et al. 1999
). The most likely difference between studies is that our DID assay utilized mice from the C57BL/6J strain which are high alcohol preferring animals; whereas Smith et.al.’s study utilized rats that needed to be given low doses of ethanol for weeks before voluntary drinking was established. Throughout the adaptation period, where rats learned to drink increasing alcohol doses that produced robust blood ethanol concentrations, they were exposed to nicotine daily. Thus, chronic nicotine enhanced ethanol consumption, while our study illustrates that acute nicotine in naïve mice reduces ethanol intake. It will be interesting to determine the effect of chronic nicotine exposure on consumption in the DID assay.
Our results indicate that cytisine can also reduce ethanol drinking. While nicotine is a full agonist, cytisine is known to be a full agonist for β4* nAChRs and a partial α4β2 agonist (Mineur et al. 2007
; Picciotto et al. 1995
). The α4β2 selective partial agonist, varenicline is a derivitave of cytisine and recently has been shown to inhibit alcohol intake and seeking in rats (Coe et al. 2005
; Steensland et al. 2007
). Based on these observations, cytisine may also be a candidate compound for alcohol cessation.
Mecamylamine and nicotine differentially modulate alcohol drinking patterns. Mecamylamine reduced ethanol intake predominantly in the second hour of the DID assay; whereas nicotine reduced intake during the first hour, perhaps indicating independent mechanisms of action for each compound. Although drinking patterns may be explained by differences in the pharmacokinetics of each drug and how readily they cross the blood brain barrier. Nicotine is known to permeate the brain on the order of seconds (Lockman et al. 2005
), while mecamylamine likely has a longer latency to reach effective concentrations in the CNS (Young et al. 2001
Because of the complexity of nAChR subunit composition, as well as the robust expression patterns of nAChRs throughout the CNS, it is not so surprising that blocking nAChRs (i.e. with mecamylamine) and activating them with agonist can both reduce ethanol intake. However, could both classes of compounds impact the same ethanol reward circuit to impact voluntary ethanol intake? Based on multiple studies indicating that nAChRs rapidly desensitize after a single nicotine exposure, often for prolonged periods of time (Mansvelder et al. 2002
; Pidoplichko et al. 1997
), it is possible that an acute injection of nicotine or cytisine prior to ethanol exposure desensitizes the relevant nAChR subtype precluding activation of circuits involved in voluntary drinking. Thus, blocking nAChRs with an antagonist or desensitizing nAChRs with pre-exposure to agonists would both reduce alcohol consumption. Our c-Fos/TH double labeling experiments support this idea. Pre-injection of mecamylamine significantly reduced the number of DAergic neurons in the VTA that were activated by a subsequent exposure to ethanol suggesting that mecamylamine may block ethanol reward.
Alternatively, ethanol intake may be reduced by the nAChR agonists because the agonists themselves elevate nucleus accumbens DA release, thereby increasing DA signaling prior to ethanol drinking (Marubio et al. 2003
; Picciotto et al. 1998
). Indeed, pre-injection of nicotine increased c-Fos induction in DAergic neurons and a subsequent exposure to ethanol did not increase c-Fos further compared to nicotine alone, suggesting that nicotine and alcohol may activate similar reward pathways. The DA reuptake blocker GBR 12909 has been shown to also reduce ethanol intake in the DID paradigm, presumably via a similar mechanism (Kamdar et al. 2007
) but this compound was also shown to decrease sugar water intake. Our results argue against a common reward pathway because nicotine and cytisine reduced ethanol intake without reducing sucrose drinking suggesting that nicotinic receptor activation is involved in alcohol/nicotine reward specifically.
In summary our data indicate that nAChRs are involved in acute ethanol drinking until intoxication. Identification of the specific nAChR subtypes involved in this behavior should lead to novel therapeutic targets that could be used to prevent binge drinking.