The main finding of the present study is that the mGluR5 antagonist MPEP decreased the reinforcing effects of ethanol in alcohol-preferring inbred C57BL6/J mice. The mGluR1 antagonist CPCCOEt or the mGluR2/3 antagonist LY 341495 were without effect on ethanol-reinforced responding. These data are consistent with emerging evidence implicating mGluR5 in the general regulation of the reinforcing effects of drugs of abuse. For example, mice lacking the mGluR5 gene do not self-administer cocaine and show no cocaine-induced increase in locomotor activity (Chiamulera et al. 2001
), which indicates a significant role of mGluR5 in the behavioral effects of psychomotor stimulants. MPEP dose-dependently reduced nicotine self-administration in rats (Paterson et al. 2003
). Recent evidence also indicates that MPEP decreases ethanol self-administration and blocks relapse to ethanol self-administration in rats (Backstrom et al. 2004
; Schroeder et al. 2005
). Together, these data suggest that mGlu5 receptor activity is required for the full expression of cocaine, nicotine, and ethanol reinforcement in both rats and mice, which demonstrates interspecies generality of mGluR5 involvement in addiction.
Results from the present study also suggest that mGluR5 modulation of ethanol reinforcement is specific. That is, MPEP decreased ethanol self-administration in the absence of effect on concurrent water-reinforced responding. This is consistent with MPEP reductions in nicotine self-administration, with no concomitant effect on food-maintained responding (Markou et al. 2004
; Paterson et al. 2003
; Tessari et al. 2004
) and evidence that mGluR5 null mice respond for food reinforcement (Chiamulera et al. 2001
). This interpretation warrants caution, however, because ethanol- and water-reinforced response rates were significantly different, which may reflect differential reinforcement function and sensitivity to mGluR5 modulation. In addition, mGluR5 antagonists decrease food-reinforced responding (Paterson and Markou 2005
; Varty et al. 2005
), and caloric content influences ethanol self-administration by C57BL/6NHsd (McMillen and Williams 1998
) and C57BL/6ByJ (Bachmanov et al. 1996a
; Bachmanov et al. 1996b
) mice, which suggests that observed effects of MPEP on ethanol self-administration may have been influenced by the caloric properties of ethanol. That said, MPEP also decreases ethanol- but not water-reinforced responding in selectively bred alcohol-preferring P rats (Schroeder et al. 2005
), which are considered a valid animal model of alcoholism because their ethanol intake does not appear to be influenced by taste or caloric factors (Lankford et al. 1991
; McMillen 1997
). Together, these data suggest that mGluR5 activity selectively regulates drug and ethanol reinforcement when alternative reinforcers are available but may also play a more general role in motivation.
In addition to effects on the total number of ethanol reinforced responses, the mGluR5 antagonist MPEP produced specific changes in the temporal distribution of responding that suggest reduced motivation to self-administer ethanol. First, MPEP (10 mg/kg) produced a 13-fold delay in the onset of the first ethanol-reinforced response, which is consistent with reduced motivation to start drinking (Schroeder et al. 2003
). Second, ethanol-reinforced responding is well known to occur in temporally contiguous units called “bouts.” This is observed in primates (Henningfield and Meisch 1979
), rats (Samson et al. 1988
), and mice (Olive et al. 2000
). In the present study, MPEP (10 mg/kg) decreased the total number of ethanol-reinforced bouts and the rate of responding during each bout. Thus, mice engaged in fewer temporally clustered units of self-administration behavior, and responding during each cluster was slower following MPEP injection. When taken together with the significant delay in response onset, these data are consistent with reduced motivation to self-administer ethanol.
However, a number of interpretations other than altered motivation are also plausible. First, MPEP may have altered ethanol-reinforced responding via nonspecific effects on motor ability. For the most part, however, locomotor data from the present study argue against this interpretation because the mGluR5 antagonist did not alter motor activity during the first 6 h of a 16-h locomotor session, which is when the compound reduced ethanol self-administration. A second alternative interpretation is that MPEP may have altered the rewarding effects of ethanol or produced independent rewarding or aversive effects that decreased ethanol self-administration. This possibility also appears unlikely because MPEP does not alter ethanol-induced conditioned place preference (CPP) or produce a CPP or conditioned place aversion when administered alone (McGeehan and Olive 2003
; Popik and Wrobel 2002
). Third, a plausible alternative explanation to altered motivation is that MPEP may have altered the pharmacological effects of ethanol that underlie its reinforcing function. Research from our laboratory has shown that MPEP blocks the discriminative (i.e., subjective) stimulus effects of ethanol (Besheer and Hodge 2005
), which are fundamental to addiction liability (Stolerman 1992
). Thus, MPEP may have blocked the discriminative stimulus effects of self-administered ethanol, which could functionally shift the concentration response curve for ethanol reinforcement to the left and occasion less responding. It would be of significant interest to determine in future studies if MPEP can alter the discriminative stimulus effects of self-administered ethanol (e.g., Hodge et al. 2001
), which would suggest a highly specific behavioral mechanism of action.
Data from the present experiment show that mGluR1 or mGluR2/3 antagonists were without effect on ethanol- or water-reinforced responding. This result is consistent with recent evidence showing that glutamate activity at mGluR5, but not mGluR1 or mGluR2/3, regulates the reinforcing effects of ethanol in selectively bred alcohol-preferring P rats (Schroeder et al. 2005
), which raises the possibility that mGluR regulation of ethanol reinforcement is specific to the mGluR5 subtype. However, reduced potency of the mGluR1 antagonist at its receptor should be considered as an alternative explanation for lack of effect of CPCCOEt. Future studies employing different antagonists, doses, or agonist compounds may find functional involvement of mGluR1 or mGluR2/3 receptors in ethanol reinforcement. At the present time, however, the results of this study suggest both reinforcer and receptor subtype specificity in the modulation of ethanol reinforcement by mGluR5.
It is of significant interest to consider why the mGluR antagonists showed differential involvement in ethanol reinforcement. Although group I mGluRs (mGluR1 and mGluR5) are highly related, these receptors have a distinct pattern of expression in the brain that may determine functional involvement in ethanol self-administration. MGluR1 receptors show low expression in most limbic brain regions but are highly expressed in the cerebellum where it regulates motor coordination (Ichise et al. 2000
). MGluR5 (and mGluR2/3) receptors are almost completely absent in the cerebellum, but are highly expressed in limbic brain regions such as the nucleus accumbens, cortex, and hippocampus, where mGluR1 receptors are less abundant (Bordi and Ugolini 1999
; Spooren et al. 2001
; Tamaru et al. 2001
). This differential mesocorticolimbic distribution pattern suggests differential involvement in ethanol self-administration. For example, evidence from site-specific infusion studies shows ethanol self-administration is modulated by the activity of specific brain regions, including the ventral tegmental area, nucleus accumbens, and frontal cortex (Hodge et al. 1995
; Rassnick et al. 1992
; Samson et al. 1993
) that express high levels of mGluR5 and mGluR2/3. Thus, based on expression patterns, it is plausible that mGlu5 receptors might specifically regulate ethanol reinforcement, but mGluR1 might not.
Given the similar expression patterns of mGlu2/3 and mGlu5 receptors, it does not seem likely that the differential effect of antagonists at these two receptors can be attributed to brain regional influence. However, evidence suggests that these receptors may be predominantly expressed at different cellular locations. For example, mGluR5 immunoreactivity is predominantly postsynaptic (Paquet and Smith 2003
), whereas mGlu2/3 receptors are mostly presynaptic (Tamaru et al. 2001
). Thus, the mGluR5 antagonist may have inhibited primarily the postsynaptic effects of glutamate (e.g., O’Leary et al. 2000
), which appeared to interfere with ethanol reinforcement. By contrast, the pharmacological blockade of presynaptic mGlu2/3 receptors increases extracellular glutamate levels (Baker et al. 2002
), which did not alter ethanol reinforcement. Therefore, the actions of glutamate at postsynaptic mGlu5 receptors appear to be necessary for full expression of ethanol reinforcement. Moreover, it appears plausible that decreasing synaptic glutamate levels via administration of an mGluR2/3 agonist might also decrease ethanol reinforcement, which has been shown to decrease reinstatement of cocaine self-administration (Baptista et al. 2004
The data from this study suggest that mGluR5 activity is required for the full expression of ethanol’s reinforcing effects. However, the long time course of MPEP effect on ethanol self-administration (i.e., up to 6 h postadministration) indicates that the observed behavioral effects are not correlated with direct pharmacological blockade of the receptor. That is, in vivo occupancy of mGluR5 by MPEP (10 mg/kg) decreases linearly to control levels after 2 h, with an approximate half-life of 1 h in C57BL/6J mice (Anderson et al. 2003
). This suggests that MPEP administration may have altered the activity of signaling pathway (s) downstream of mGluR5 that inhibited ethanol self-administration during the effective period of 3–6 h postadministration. Indeed, activation of postsynaptic mGluR5 up-regulates phospholipase C (PLC) activity, which in turn activates the second messenger diacylglycerol (DAG) (Linn 2000
; Pellegrini-Giampietro et al. 1996
). DAG activates protein kinase C (PKC), which can increase Ca2+
influx via phosphorylation of ionotropic NMDA receptors (MacDonald et al. 1998
), which influence ethanol intake (McMillen et al. 2004
). Accordingly, we have shown that gene deletion of PKCε, which is DAG-sensitive, decreases ethanol self-administration (Hodge et al. 1999
), and that the effects of MPEP on two-bottle ethanol intake are dependent on PKCε (Olive et al. 2005
). Together, these findings suggest that MPEP administration may produce long-term reductions in alcohol self-administration by changing the function of mGluR5 coupled signaling pathways.
In conclusion, many studies have focused on iGluR involvement in ethanol’s neurobiological effects, but the clinical utility of manipulating iGluRs remains illusive due to numerous side effects associated with their widespread central nervous system (CNS) expression (Kemp and McKernan 2002
). MGluRs are rapidly emerging as important therapeutic targets in numerous areas of neuropathology (Bordi and Ugolini 1999
; Spooren et al. 2001
), but few studies have addressed their involvement in ethanol’s reinforcing effects. The results of this preclinical study indicate that the mGluR5 antagonist MPEP decreases the reinforcing effects of ethanol and may therefore have utility in the medical management of alcohol abuse and alcoholism, insomuch as reduced drinking is a viable therapeutic outcome.