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- Materials and methods
Alcohol-induced increases in nucleus accumbens glutamate actively regulate alcohol consumption, and the alcohol responsiveness of corticoaccumbens glutamate systems relates to genetic variance in alcohol reward. Here, we extend earlier data for inbred mouse strain differences in accumbens glutamate by examining for differences in basal and alcohol-induced changes in the striatal expression of glutamate-related signaling molecules between inbred C57BL/6J and DBA2/J mice. Repeated alcohol treatment (8 × 2 g/kg) increased the expression of Group1 metabotropic glutamate receptors, the NR2a/b subunits of the N-methyl-d-aspartate receptor, Homer2a/b, as well as the activated forms of protein kinase C (PKC) epsilon and phosphoinositol-3-kinase within ventral, but not dorsal, striatum. Regardless of prior alcohol experience, C57BL/6J mice exhibited higher accumbens levels of mGluR1/5, Homer2a/b, NR2a and activated kinases vs. DBA2/J mice, whereas an alcohol-induced rise in dorsal striatum mGluR1/5 expression was observed only in C57BL/6J mice. We next employed virus-mediated gene transfer approaches to ascertain the functional relevance of the observed strain difference in accumbens Homer2 expression for B6/D2 differences in alcohol-induced glutamate sensitization, as well as alcohol preference/intake. Manipulating nucleus accumbens shell Homer2b expression actively regulated these measures in C57BL/6J mice, whereas DBA2/J mice were relatively insensitive to the neurochemical and behavioral effects of virus-mediated changes in Homer2 expression. These data support the over-arching hypothesis that augmented accumbens Homer2-mediated glutamate signaling may be an endophenotype related to genetic variance in alcohol consumption. If relevant to humans, such data pose polymorphisms affecting glutamate receptor/Homer2 signaling in the etiology of alcoholism.
- Top of page
- Materials and methods
This report describes differences in NAC glutamate receptor-associated protein expression between two inbred mouse strains, C57BL/6J (B6) and DBA2/J (D2) mice, which are highly utilized for studying the neurogenetics of alcoholism (Crabbe 2002; Phillips 1997). Consistent with the existing immunoblotting data for alcohol drinking (Cozzoli et al. 2009; Szumlinski et al. 2008), repeated treatment of both strains with a sensitizing alcohol injection regimen elevated NAC expression of Group1 mGluR subtypes and their associated scaffolding protein Homer2 (c.f., Shiraishi-Yamaguichi & Furuichi 2007). Also consistent with the previous alcohol-drinking studies conducted in both mice (Cozzoli et al. 2009; Szumlinski et al. 2008) and P rats (Obara et al. 2009), no alcohol-induced changes in striatal Homer1b/c expression were observed and the majority of alcohol-induced changes in glutamate receptor/Homer2 levels were selective for the more ventral vs. dorsal striatum.
In the present injection study, the alcohol-induced changes in protein expression were more, rather than less, consistent between the two NAC subregions. These data extend earlier evidence for a hyper-responsiveness of NAC post-synaptic glutamate receptor signaling during early alcohol withdrawal (Cozzoli et al. 2009; Szumlinski et al. 2005, 2008) and indicate that repeated, behaviorally non-contingent, alcohol treatment augments Group1 mGluR/Homer2-mediated signaling within both the ‘motor’ (NAC core) and ‘motive’ (NAC shell) subcircuits of the limbic system (for recent reviews, Everitt et al. 2008; Meredith et al. 2008). Interestingly, in a recent study conducted in chronic (6 months) alcohol-drinking P rats, effects of alcohol intake were observed upon Group1 mGluR, NR2 and Homer2 protein expression in the NAC core subregion only (Obara et al. 2009). Such data are consistent with earlier immunoblotting data from cocaine studies, indicating that Homer-related signaling is activated within different ventral striatal subcircuits by self-administered vs. experimenter-administered drug, with the more dorsal core region exhibiting changes in self-administering, but not experimenter-injected animals (Ary & Szumlinski 2007; Ben-Shahar et al. 2009). Whether or not the apparent subregional differences in the effects of self-administered alcohol (Obara et al. 2009) vs. experimenter-injected alcohol (Cozzoli et al. 2009; Szumlinski et al. 2008) relate to the route of drug administration, the duration of alcohol exposure, other non-pharmacological factors associated with drug administration (e.g. stress of injection, predictability of alcohol effects/expectancy or conditioned factors) or the species employed cannot be discerned at the present time. Nevertheless, such data are consistent with current addiction theories that habitual drug-taking behavior is associated moreso with the activation of more dorsal striatal structures (Everitt et al. 2008; Kalivas & McFarland 2003; Meredith et al. 2008).
Alcohol exposure or re-exposure to alcohol-associated cues is reported to elevate the activational state, translocation or total protein expression of PKCɛ, PI3K and ERK (Cozzoli et al. 2009; Messing et al. 1991; Olive et al. 2005; Schroeder et al. 2008; but see Kumar et al. 2006). In contrast to these earlier results, but consistent with a report indicating a tolerance to the effects of alcohol-induced PKCɛ translocation with repeated alcohol treatment (Kumar et al. 2006), we failed to detect the effects of repeated alcohol treatment upon either total kinase expression or indices of kinase activation at 24 h following the last alcohol injection. Thus, in contrast to repeated bouts of excessive alcohol consumption (∼1.5 g/kg alcohol in 30 min/day for 7 days; Cozzoli et al. 2009) or alcohol-associated cues in rats with a history of response-contingent oral alcohol intake (Schroeder et al. 2008), repeated, experimenter-administered injections of 2 mg/kg alcohol do not elicit an enduring increase in the constitutive activity of these downstream mediators of glutamate receptor signaling, at least when assessed by total protein expression. These discrepant results might relate to the very distinct alcohol delivery procedures employed between the injection and self-administration studies, implicating differences in alcohol pharmacokinetics and/or non-pharmacological factors associated with alcohol delivery (i.e. motivation, stress, control over intake, anticipation and associative learning) in the pattern or duration of alcohol-induced kinase activation within the NAC.
A positive relation exists between a genetic propensity to consume high amounts of alcohol and basal NAC mGluR/Homer2/kinase activity, as assessed by comparing between selectively bred mouse lines and between wild-type and mGluR5 mutant mice that differ in alcohol intake under scheduled, limited-access conditions (Cozzoli et al. 2009). Consistent with these ealier data, alcohol-preferring B6 mice exhibit higher indices of activated PI3K and PKCɛ in the NAC compared with alcohol-avoiding D2 mice. Moreover, the strain differences in basal kinase activity were positively associated with a parallel divergence in basal Group1 mGluR and Homer2 expression. As (1) Group1 mGluRs and Homer2 regulate alcohol-induced glutamate release within the NAC (Lominac et al. 2006; Szumlinski et al. 2005, 2008) and (2) pronounced B6/D2 differences exist in the effects of repeated alcohol administration upon NAC extracellular glutamate (Fig. 6; Kapasova & Szumlinski 2008), one might also expect that repeated alcohol treatment would sensitize glutamate receptor/Homer2-mediated signaling in B6 mice, whereas D2 mice would exhibit either no change in, or a tolerance to, alcohol-induced signaling upon repeated alcohol treatment. Although inspection of the NAC shell data for mGluR5, NR2b and the p-PKCɛ:PKCɛ ratio indicated trends that support for this hypothesis, significant alcohol-dependent strain differences in protein expression were not observed for the NAC shell. Such data indicate that the differential behavioral sensitivity to injected alcohol observed between B6 and D2 mice (Cunningham et al. 1992; Phillips 1997; Phillips et al. 1994) might relate moreso to strain differences in basal post-synpatic glutamate signaling, rather than in the responsiveness of this signaling to repeated treatment with moderate doses of alcohol (i.e. 2 g/kg). Although D2 mice exhibit a shift to the left relative to B6 mice in the dose–response functions for both alcohol-induced locomotor sensitization and conditioned place-preference (Camarini et al. 2000; Cunningham et al. 1992; Lessov et al. 2001; Metten et al. 1998; Phillips 1997; Phillips et al. 1994), repeated treatment with 2 g/kg alcohol is capable of eliciting locomotor sensitization and a conditioned place-preference in B6 mice (Lominac et al. 2006; McGeehan & Olive 2003; Szumlinski et al. 2008, but see Camarini & Hodge 2004). NAC Homer2 over-expression in B6 mice can promote the locomotor-sensitizing and conditioned rewarding properties of injected alcohol (Szumlinski et al. 2008). In this study, the magnitude of the rise in NAC mGluR/NR2/Homer2 expression and kinase activity elicited by repeated treatment with 2 g/kg alcohol was more rather than less strain-independent. From such observations, it is suggested that certain forms of alcohol-induced behavioral plasticity may be mediated by a relative increase in NAC glutamate signaling with repeated alcohol experience, rather than the absolute amount of Homer-mediated signaling through Group1 mGluRs and NMDA receptors following alcohol experience.
Although the average baseline glutamate values obtained in this study are lower than those reported in some of our previous reports (a finding that very likely reflects variance in probe recovery/construction), consistent with the results of our previous neurochemical comparison of B6 vs. D2 mice (Kapasova & Szumlinski 2008), strain differences in NAC baseline glutamate were not observed in alcohol-naÏve mice, suggesting that basal glutamate tone within the NAC is not probably a major contributing factor to the divergent alcohol phenotypes of these two inbred mouse strains. Repeated treatment with 1 g/kg alcohol is reported to elevate NAC extracellular glutamate levels in rats (Melendez et al. 2005) and we reported previously that strain differences exist with respect to the capacity of a repeated 2 g/kg alcohol injection regimen (identical to that employed in the present study) to elevate NAC baseline glutamate levels, with B6, but not D2, mice exhibiting this form of neurochemical plasticity (Kapasova & Szumlinski 2008). However, in the present study, we failed to detect an alcohol-induced rise in NAC glutamate content in B6 mice (Table 1), a finding that is consistent with the results of two other reports from our laboratory demonstrating no significant effect of repeated alcohol injection (at either 2 or 3 g/kg alcohol) upon indices of basal NAC glutamate content in either inbred B6 or B6–129 hybrid mice (Szumlinski et al. 2005, 2008). Interestingly, the two studies in the literature indicating an effect of repeated alcohol injection upon NAC basal glutamate content were conducted in virus-naÏve animals (Kapasova & Szumlinski 2008; Melendez et al. 2005), whereas the negative results in the present study (Fig. 6), as well as those previous from our group (Szumlinski et al. 2005, 2008), were derived from AAV-infused mice. This raises the possibility that our AAVs may be interfering somehow with the development of this specific alcohol-induced neurochemical adaptation in B6 animals. However, arguing against this possibility, repeated bouts of binge alcohol drinking (∼1.5 g/kg/day, every third day for a total of 6 days) do not elevate basal NAC glutamate levels in AAV-naÏve B6 mice (Szumlinski et al. 2007). Alternatively, the discrepancies in findings across studies might be attributable to differences in probe recovery; however, our earlier studies and that conducted by Melendez et al. (2005) all confirmed the presence or absence of alcohol effects in follow-up experiments employing the no net-flux technique, which is independent of probe recovery (Parsons & Justice, 1994 for discussion). Thus, inconsistencies across data sets do not appear to be reflect any obvious issues associated with differential probe recovery. Alternatively, localization of the microdialysis probes within the NAC may be a contributing factor to account for the presence vs. absence of an alcohol effect upon basal NAC glutamate content. In this study, we included data from all animals exhibiting neuronal transfection within the boundaries of the NAC, irrespective of their subregional localization, whereas in our previous strain difference study, data were included only for animals exhibiting placements within the NAC shell subregion (Kapasova & Szumlinski 2008). However, arguing against this case, our probe placements were localized primarily to the NAC shell in our previous AAV studies of B6 and B6–129 hybrid mice (Szumlinski et al. 2005, 2008), whereas those in the rat study by Melendez et al. (2005) did not discriminate between NAC subregions. Thus, at the present time, it is difficult to discern precisely why the effects of repeated alcohol injection upon basal NAC glutamate levels are inconsistent across studies. However, the fact that such inconsistencies exist supports a lesser role for this particular neurochemical adaptation in mediating not only strain differences in alcohol behavioral sensitivity, but also the effects of repeated alcohol administration upon the subsequent behavioral and neurochemical responsiveness to alcohol within a particular strain.
As we reported previously (Kapasova & Szumlinski 2008), D2 mice exhibit a large rise in NAC glutamate in response to an acute injection of 2 g/kg alcohol, whereas an acute alcohol injection or an acute bout of alcohol drinking elicits a very modest or no rise in B6 animals (Fig. 6a; Kapasova & Szumlinski 2008; Szumlinski et al. 2007, 2008). Our earlier data indicate a necessary role for both Group1 mGluRs and Homer2 for acute alcohol-induced glutamate release within the NAC (Lominac et al. 2006; Szumlinski et al. 2005), although the mechanism(s) through which this occurs is elusive, given the post-synpatic localization of Homer proteins (Shiraishi-Yamaguchi & Furuichi 2007). It is interesting to note that inbred mouse strain differences in the NAC glutamate response to acute alcohol appear to be inversely related to NAC glutamate receptor/Homer2/kinase expression (Table 2). Such discrepancies in findings point to genetic variance in other regulators of NAC extracellular glutamate/glutamate release (e.g. Group II mGluR autoreceptors, excitatory amino acid transporters and the cystine–glutamate exchanger; see Baker et al. 2002; Melendez et al. 2005) to account for the divergent acute alcohol glutamate phenotypes between B6 and D2 mice. Although it remains to be determined whether or not the large glutamate response to an acute injection of alcohol observed in D2 mice reflects some general hypersensitivity of NAC glutamatergic terminals, the fact that this strain exhibits lower expression of glutamate receptors/Homer2 suggests that this may be the case. That is to say, the lower glutamate receptor/Homer2 expression exhibited by D2 mice reflects a compensatory downregulation in post-synaptic aspects of glutamate transmission within the NAC of this strain. This hypothesis and the relevance of such neuroadaptations to the increased sensitivity of D2 mice to the acute psychomotor-activating properties of alcohol (Phillips 1997) will be topics of future studies.
Table 2. Comparison of strain differences in NAC protein expression, alcohol-stimulated glutamate release and measures of alcohol reward between control (Scrambled) B6 and D2 mice
|Basal and alcohol-induced changes in protein expression|
| Homer2a/b||B6 > D2|
| mGluR1||B6 > D2|
| mGluR5||B6 > D2|
| NR2b (only following alcohol Rx)||B6 > D2|
| PKCɛ activation||B6 > D2|
| PI3K activation||B6 > D2|
|NAC extracellular glutamate|
| Basal content||B6 = D2|
| Acute alcohol-induced release||B6 < D2|
| Repeated alcohol-induced release||B6 > D2|
| Alcohol preference||B6 > D2|
| Alcohol intake||B6 > D2|
| Water intake||B6 < D2|
However, consistent with an active and necessary role for NAC Homer2 expression in the development of alcohol-induced NAC glutamate sensitization (Szumlinski et al. 2005, 2008), a positive relation exists between B6/D2 strain differences in repeated alcohol-induced NAC glutamate sensitization (Kapasova & Szumlinski 2008) and basal NAC total protein expression of Group1 mGluRs/NR2b/Homer2/activated PI3K and PKCɛ (Table 2). Not precluding a role for the other major molecular regulators of extracellular glutamate mentioned above, such data compliment earlier correlative evidence obtained from studies of binge alcohol-drinking mice (Cozzoli et al. 2009; Szumlinski et al. 2007), as well as significant associations between polymorphisms in the p85α subunit of PI3K with risky alcohol-drinking behavior in human adolescents (Desrivieres et al. 2008), and further one of the working hypotheses of our laboratory that idiopathic increases in mGluR/Homer2-mediated signaling within the NAC facilitates the development of glutamate sensitization, which in turn increases the propensity for subsequent alcohol consumption.
Indeed, activity within glutamate receptor/Homer2/PKCɛ/ PI3K signaling cascades is necessary for the full expression of alcohol intake in rodents with high or moderate alcohol-consuming phenotypes (Backstrom & Hyytia 2004; Besheer et al. 2006; Choi et al. 2002; Cozzoli et al. 2009; Hodge et al. 2006; Lominac et al. 2006; Newton & Messing 2007; Olive et al. 2000, 2005; Schroeder et al. 2005; Szumlinski et al. 2005, 2008), a finding supported further by the present behavioral and neurochemical data for shRNA- and cDNA-treated B6 mice (Table 3). Moreover, immunoblotting data obtained from inbred, selectively bred and transgenic mouse models of genetic variability in alcohol intake provide congruent, albeit correlative, evidence for a link between variance in basal NAC glutamate receptor/Homer2/PKCɛ/PI3K signaling and alcohol preference/intake (Figs 3 and 4; Cozzoli et al. 2009). Although strain differences in alcohol intake/preference between B6 and D2 mice have been attributed to strain differences in taste reactivity (Blizard 2007; McClearn & Rodgers 1959), D2 mice exhibit less operant behavior for intravenous alcohol reinforcement than do B6 mice (Grahame & Cunningham, 1997), indicating that strain differences in mechanisms other than taste reactivity are also contributing to the alcohol-avoiding/low preference phenotype of D2 mice. In further support of this suggestion, an intermittent alcohol injection regimen, akin to that demonstrated presently to elevate NAC mGluR/NR2/Homer2 expression and kinase activation, can promote alcohol consumption in D2 mice (Camarini & Hodge 2004). These latter data suggest that repeated experience with injected alcohol can elicit some form of neuroplasticity that promotes drinking behavior that does not relate directly to orosensory experience. That being said, the rostrocaudal portions of the NAC regulate taste hedonics in rodents and glutamate transmission appears to play a role in this regard (Dourish et al. 1986; Kelley & Swanson 1997; Maldonado-Irizarry et al. 1995; Reynolds & Berridge 2002). As NAC Homer2 over-expression augments free-access alcohol intake in both high alcohol-preferring B6 mice (Szumlinski et al. 2008), as well as more moderate alcohol-drinking B6–129 hybrid mice (Szumlinski et al. 2005) and shRNA-mediated knockdown of NAC Homer2 expression attenuates binge alcohol drinking in B6 mice (Cozzoil et al. 2009), it was hypothesized that if reduced basal NAC shell Homer2 expression actively contributed to the alcohol-avoiding profile of D2 mice (by affecting taste hedonics, general reward mechanisms or both), then Homer2 over-expression in this region (i.e. mimicking the effects of injected alcohol) should promote higher alcohol intake/preference in D2 mice, whereas Homer2 knockdown might further reduce these indices of alcohol reward in this strain. However, our data clearly show that neither AAV construct significantly modified alcohol drinking by D2 mice, despite robust neuronal transfection (Figs 1 and 2) and despite producing the predicted effects in B6 animals (Fig. 7). Interestingly, the strain differences in sensitivity to AAV-mediated changes in NAC Homer2 expression are paralleled by the present data for alcohol-induced glutamate sensitization (Table 3) and are more or less consistent with the results of a recent pharmacological study in which manipulating NAC shell extracellular glutamate levels was found to actively regulate alcohol drinking in B6 mice, but not in D2 mice (Kapasova & Szumlinski 2008). One very plausible explanation for the apparent behavioral and neurochemical insensitivity of D2 mice to NAC glutamate/Homer2 manipulations could relate to their relatively low glutamate receptor expression, the functional consequences of which (i.e. taste hedonics or reward/reinforcement) may not be overcome by simply increasing the amount of a downstream mediator-like Homer2. Unfortunately, AAV or lentiviral vectors carrying cDNA for Homer2-associated glutamate receptors (mGluR1/5, NR2) are not currently available to test this hypothesis directly. However, an alternate route of investigation might involve extending the present results to include other inbred mouse strains that exhibit not only high and low, but also intermediate alcohol-drinking phenotypes, to better correlate genetic variance in intake with basal/alcohol-stimulated changes in mGluR/NR2/Homer2/kinase signaling, as well as the effects of NAC Homer2 manipulations upon measures of alcohol reward.
Table 3. Comparison of the effects of AAV-mediated increases and decreases in NAC Homer2 expression upon strain differences in alcohol-induced NAC glutamate release and alcohol-drinking behavior
|Acute alcohol-induced glutamate release||+||nc||nc||−|
|Repeated alcohol-induced glutamate release||+||−||nc||nc|