Binge alcohol drinking co-regulates NAC Homer2 and NR2 expression, as well as PI3K activation in B6 mice
To extend our earlier immunoblotting data from an animal model of moderate alcohol intake (Szumlinski et al., 2008b
) to one of excessive, binge alcohol drinking (i.e., BACs>0.8 mg/ml; NIAAA, 2007
), we first examined the effects of 6 bouts of SHAC upon the total protein expression of Homer2a/b, Homer1b/c, their associated glutamate receptors (mGluR1, mGluR5, NR2a, NR2b), PI3K, as well as the p(Tyr)p85α PI3K binding motif, within several brain regions implicated in the neurobiology of alcoholism, including the NAC, prefrontal cortex (PFC), striatum and hippocampus (e.g., Sullivan and Pfefferbaum, 2005
; Bell et al., 2006
; Koob and Le Moal, 2008
). The B6 mice exposed to the SHAC procedure in this experiment exhibited a mean alcohol intake of 1.6 ± 0.2 g/kg over the 6, 30-min alcohol bottle presentations, which (based on the results of our regression analysis; ) was predicted to yield a BAC of 1.03 mg/ml and demonstrated previously by our group to result in an actual BAC of 1.09 ± 3.73 mg/ml (Szumlinski et al., 2007
). These data indicate (1) the feasibility of employing the results of our regression analysis to predict BACs from observations of alcohol intake in our studies and (2) by NIAAA standards (NIAAA, 2007
), the experimental mice in the immunoblotting study exhibited binge alcohol consumption under our SHAC procedures.
As summarized in and , binge alcohol drinking co-regulated Homer2a/b, NR2a/b and p(Tyr)p85α PI3K binding motif levels selectively within the NAC. Consistent with the effects of continuous alcohol drinking (Szumlinski et al., 2008b
), binge drinking under the SHAC procedure more than doubled NAC Homer2a/b expression (t23
=2.22, p=0.04), without affecting significantly Homer1b/c levels (). Moreover, the SHAC procedure-induced rise in NAC Homer2a/b was accompanied by a smaller, albeit significant, increase in NAC levels of NR2a (t23
=2.35, p=0.03) and NR2b (t23
=2.75, p=0.01), but binge drinking under the SHAC procedure did not increase the total protein expression of either Group 1 mGluR subtype (). Consistent with the hypothesis that excessive alcohol drinking induces the activation of PI3K within the NAC, B6 mice exposed to the SHAC procedure exhibited increased p(Tyr)p85α PI3K binding motif levels (t24
=2.34, p=0.03), without any change in total PI3K expression.
Figure 3 Repeated bouts of binge alcohol intake with the SHAC procedure elevate NAC protein expression of Homer2, NR2 subunits and PI3K activation in B6 mice. a, Representative immunoblots for the total protein levels of Homer2a/b, Homer1b/c, mGluR1, mGluR5, NR2a, (more ...)
Table 1 Summary of the means ± SEM of the effects of 6 bouts of 30-min access to 5% alcohol upon Homer1b/c, Homer2a/b, mGluR1, mGluR5, NR2a, NR2b, and PI3K within the prefrontal cortex (PFC), dorsal striatum and hippocampus of mice. The data are expressed (more ...)
In contrast to the data for the NAC (), binge alcohol drinking under the SHAC procedure failed to significantly affect Homer/mGluR/NR2/PI3K protein expression or the levels of p(Tyr)p85α PI3K binding motif within the dorsal striatum. Of all the proteins examined, only expression of NR2b increased within the PFC (). However, as summarized in , binge alcohol drinking under the SHAC procedure increased the hippocampal protein expression of mGluR1a (t25=2.62, p=0.02) and mGluR5 (t27=2.24, p=0.03), as well as NR2b (t25=3.01, p=0.01), but did not affect the expression of the other proteins examined. These data demonstrate for the first time that a history of binge alcohol drinking promotes PI3K signaling selectively within the NAC, which appears to be associated with a rise in Homer2a/b, but not Group1 mGluR, expression.
Reduction in binge alcohol drinking by NAC Homer2 knock-down
Homer2 expression within the NAC shell actively regulates alcohol intake under continuous access and operant self-administration conditions (Szumlinski et al., 2005b
). As the SHAC procedure elicited a large rise in the NAC total protein expression of Homer2a/b (), the first series of behavioral experiments employed an AAV-mediated neuronal transfection strategy (see Klugmann and Szumlinski, 2008
for review) to examine the functional relevance of the SHAC-induced rise in NAC Homer2 protein expression for excessive, binge alcohol drinking behavior. As reported previously (Szumlinski et al., 2004
; Klugmann et al., 2005
; Lominac et al., 2005
), immunostaining for the HA tag revealed neuronal transfection for all three AAVs employed (cDNA-Homer2b, shRNA1-Homer2b, shRNA2-Homer2b) with no overt signs of neurotoxicty, regardless of vector or the localization of the transfection. Consistent also with our previous reports and as exemplified by and its subpanels (target = NAC core), transfection was observed within both cell bodies and processes within 1 mm of the infusion site (″).
In contrast to the data obtained using other animal models of alcohol reward/intake (Szumlinski et al., 2005b
), AAV-mediated Homer2b over-expression within the NAC shell failed to enhance significantly the alcohol intake exhibited by B6 mice with the SHAC procedure, despite the average intakes of both AAV treatment groups being ~0.75 g/kg over the course of 6 alcohol bottle presentations (). As illustrated in , NAC shell Homer2b over-expression also did not affect water intake under SHAC procedures, a finding consistent with our earlier reports that NAC Homer2 expression does affect the motivational valence of natural rewards (Szumlinski et al., 2004
). To examine the possibility that the effects of NAC Homer2b over-expression upon drinking with the SHAC procedure might be subregion-specific, the experiment was replicated infusing the AAVs into the NAC core. Again, we failed to detect any significant effect of NAC core Homer2b over-expression upon either alcohol intake () or water intake (). These data indicate that elevating NAC Homer2b expression in an animal with a history of binge alcohol drinking is not sufficient to enhance further their alcohol consumption, at least within the confines of the murine SHAC model.
Studies of Homer2
knock-out (KO) mice indicated that NAC Homer2 expression is necessary for alcohol consumption as assessed under both free-access and operant self-administration paradigms (Szumlinski et al., 2005b
). To avoid developmental confounds associated with the use of constitutive gene knock-out animals (e.g., Gerlai, 2001
) yet extend our earlier Homer2
KO data to an animal model of binge alcohol drinking, we employed an AAV-shRNA infusion strategy, demonstrated previously to reduce NAC shell Homer2b expression by 50% (Klugmann and Szumlinski, 2008
). The infusion of two different shRNA-Homer2b constructs into the NAC shell significantly reduced the average amount of alcohol consumed by the B6 mice during the SHAC procedure by approximately 20% () [F(2,28)=3.57, p=0.04] and post-hoc analysis indicated no differences between the two shRNA constructs in this regard. While BACs were not determined in this study, alcohol dose is highly predictive of BACs in the SHAC model (Cronise et al., 2005
; Finn et al., 2005
). Based on the results of our regression analysis (), the ~ 1 g/kg dose of alcohol consumed by shRNA-treated mice is predicted to yield a BAC of ~ 0.87 mg/ml, indicating that NAC shell Homer2b knockdown significantly attenuated, but did not prevent, the expression of binge drinking. Consistent with our earlier data for Homer2
KO mice (Szumlinski et al., 2004
), NAC shell Homer2b knock-down did not affect the average water intake of the mice exhibited during testing (), indicating that this manipulation did not influence the motivation for, or physical capacity to, consume fluids. Taken altogether, these data indicate that the large rise in NAC Homer2b expression produced by repeated bouts of binge alcohol drinking, while not sufficient, may be necessary for the full expression of excessive drinking behavior.
Reduction in binge alcohol drinking by NAC mGluR5 and PI3K blockade
Systemic pretreatment with Group1 mGluR antagonists, particularly against the mGluR5 subtype, reduces alcohol intake under both limited and continuous access conditions (e.g., Backström et al., 2004
; Olive et al., 2005
; Schroeder et al., 2005
; Hodge et al., 2006
; Lominac et al., 2006
; Bäckström and Hyytiä, 2007
; Besheer et al., 2008a
; Blednov and Harris, 2008
). While NAC mGluR5 activity is required for cue-induced cocaine-seeking behavior in rats (Bäckström and Hyytiä, 2007
), the brain regions involved in mGluR1/5 regulation of alcohol intake remain uncharacterized. Homers interact directly with the C-terminus of Group1 mGluRs (Tu et al., 1998
; Xiao et al., 1998
) and form a complex with PIKE-L, which can regulate both constitutive and stimulated PI3K activity (Rong et al., 2003
). The observed SHAC procedure-induced increases in the amount of p(Tyr)p85α PI3K binding motif expression (), suggested an important role for mGluR-mediated stimulation of PI3K activity within the NAC in maintaining binge alcohol drinking behavior. To test this hypothesis, groups of B6 mice were trained to binge drink alcohol (1.5-1.6 g/kg in 30 min; predicted BACs ~ 1.0 mg/ml; ) and then pretreated intra-NAC shell with several doses of the selective mGluR5 antagonist MPEP or a PI3K-selective dose of wortmannin (Rong et al., 2003
) prior to a 30-min alcohol drinking test session. For comparison, the effects of intra-NAC pretreatment with the selective mGluR1 antagonist CPCCOEt were also assessed.
Figure 5 Blockade of NAC mGluR5 and PI3K, but not mGluR1, reduces binge alcohol drinking in B6 mice. a, Summary of the effects of an intra-NAC shell infusion of various doses of the mGluR5 antagonist MPEP (solid bars), 50 ng/side wortmannin and the combination (more ...)
As illustrated in , alcohol intake was reduced dose-dependently by intra-NAC MPEP (0, 0.1, 0.3 and 1.0 μg/side) [F(3,27)=5.75, p=0.004], with estimated BACs of 0.99, 0.87, 0.84 and 0.83 mg/ml, respectively. An attenuation of alcohol intake was observed also following the local infusion of 50 ng/side wortmannin (estimated BACs, 0 ng wortmannin = 1.03 mg/ml; 50 ng wortmannin = 0.83 mg/ml) and the co-infusion of 50 ng/side wortmannin with 1.0 μg/side MPEP failed to further reduce alcohol intake [; F(2,10)=5.77, p=0.02]. Thus, NAC mGluR5 signaling through PI3K appears to be involved in the expression of binge alcohol drinking behavior under the SHAC procedure.
In contrast to the effect of mGluR5 and PI3K blockade, mGluR1 antagonism by intra-NAC CPCCOEt (0, 1.0 and 3.0 μg/side) produced only a moderate reduction in alcohol intake with the SHAC procedure, which failed to reach statistical significance (). Unfortunately, due to solubility issues, the effects of higher CPCCOEt doses could not be assessed. Thus, it remains to be determined whether or not the inhibitory effect of systemic pretreatment of mGluR1 selective antagonists upon alcohol intake (Lominac et al., 2006
; Besheer et al., 2008b
) involves inhibition of mGluR1 receptors within the NAC shell.
As was observed for NAC Homer2b knock-down () and consistent with published studies indicating that systemic mGluR5 blockade or mGluR5
deletion does not reduce the motivation for, or consumption of, natural reinforcers like water and sucrose nor do these pretreatments alter spontaneous locomotor activity (e.g., Backström et al., 2004
; Olive et al., 2005
; Schroeder et al., 2005
; Hodge et al., 2006
; Lominac et al., 2006
; Bäckström and Hyytiä, 2007
; Besheer et al., 2008a
; Blednov and Harris, 2008
), intra-NAC pretreatment with 1.0 μg/side MPEP or 50 ng/side wortmannin did not affect water intake during a 30-min test session (). These results indicate that neither pretreatment produced an effect upon general motivational or motor processes that could have negatively affected the ability to consume fluids under these limited-access conditions.
Reduction in binge alcohol drinking by disrupting mGluR5-Homer interaction
The capacity of mGluR5 to regulate PI3K activity in vitro
requires Homer interactions with the C-terminus of the receptor (Rong et al., 2003
). Thus, we next tested the hypothesis that the physical interaction between mGluR5 and Homer is critical for the maintenance of binge alcohol drinking behavior. For this, the SHAC phenotype of transgenic (TG) mice with a F1128R point mutation in mGluR5 (mGluR5F1128R
) that reduces Homer binding to the receptor (Tu et al., 1998
) was assessed. As observed in inbred B6 mice ( and ; see also Szumlinski et al., 2007
), our SHAC procedures elicited high levels of alcohol intake also in WT B6-129 hybrid mice (~1.5 g/kg/30 min; estimated BAC = 1.0 mg/ml), which was reduced by ~ 75% in littermate mGluR5F1128R
mutants [WT: 1.46 ± 0.24 g/kg vs. TG: 0.37 ± 0.08 g/kg; F(1,20)=14.80, p=0.001] and predicted to result in an estimated BAC of 0.69 mg/ml. As this estimate is well below the NIAAA criterion for binge drinking (NIAAA, 2007
), these genotypic differences indicate that the physical interaction between mGluR5 and Homers is necessary for maintaining binge alcohol drinking.
In contrast to the pronounced effect of the mGluR5F1128R mutation upon alcohol intake, genotypic differences were not observed for the average water intake (WT: 36.1 ± 6.87 ml/kg vs. TG: 30.1 ± 6.65 ml/kg; p>0.05) or average intake of a palatable 3% sucrose solution (WT: 1.84 ± 0.42 g/kg vs. TG: 1.87 ± 0.38 g/kg, p>0.05) exhibited by the mice during SHAC procedures.
To determine whether or not the blunted SHAC phenotype of mGluR5F1128R mutants extended to a model of voluntary alcohol preference drinking, a separate group of WT and mutant littermates were allowed free-access to 4 sipper tubes containing 0, 3, 6 and 12% alcohol (v/v) simultaneously in the home cage and drinking was monitored over a 24-hr period for a total of 3 weeks. In stark contrast to the findings with the SHAC procedure, no genotypic differences were observed either for total daily alcohol intake (WT: 10.84 ± 2.26 g/kg vs. TG: 14.00 ± 1.80 g/kg; p>0.05), total water intake (WT: 97.07 ± 22.29 ml/kg vs. TG: 87.49 ± 15.36 ml/kg; p>0.05) or alcohol preference (Genotype × Alcohol Concentration ANOVA, p>0.05) in this 4 bottle-choice, continuous access procedure. Thus, it does not appear that the reduction in binge alcohol drinking with the SHAC procedure observed in mGluRF1128R mutants reflects either a general disruption in the capacity to drink moderate amounts of alcohol. As both genotypes showed concentration-dependent patterns of voluntary alcohol intake [Alcohol effect: F(2,36)20.01, p<0.0001] and of preference [Alcohol effect: F(3,54)=3.28, p=0.03], the mGluR5F1128R mutants clearly can distinguish between the taste of the various ethanol solutions as well as WT animals. These data for (1) water and sucrose consumption under SHAC procedures and (2) alcohol/water preference and consumption under continuous access procedures, combined with unpublished behavioral phenotyping data indicating no consistent effects of the mGluR5 mutation upon various measures of emotionality, motor function and cognitive processing, all indicate that the reduction in binge alcohol drinking produced by the disruption of mGluR5-Homer interactions does not reflect non-specific effects of the mutation upon general reward, motivational, emotional or motor processes.
mGluR5-Homer binding is necessary for the “anti-binge drinking” effect of NAC mGluR5 and PI3K blockade
To test the hypothesis that signaling through an mGluR5-Homer2-PI3K pathway is critical for excessive, binge alcohol drinking, we next assessed the capacity of intra-NAC infusions of 1.0 μg/side MPEP and 50 ng/side wortmannin to reduce “binge drinking” with the SHAC procedure in WT and mGluR5F1128R littermates. Replicating the data presented above, marked genotypic differences in binge drinking were observed in vehicle-infused mice, with the intake of the mutants estimated to yield a BAC of 0.8 mg/ml versus 1.0 mg/ml in WT animals. In contrast to vehicle-infused animals, genotypic differences in binge drinking were not observed in mice pretreated intra-NAC with either MPEP or wortmannin () [Genotype effect: F(1,11)=4.32, p=0.05; Drug effect: F(2,22)=4.50, p=0.02]. The data in suggested that the effect of intra-NAC antagonist infusion was selective for WT animals, which was confirmed by the results of one-way ANOVAs conducted separately for each genotype [WT: F(2,12)=3.97, p=0.04, post-hoc tests; KO: p=0.29]. Taken all together, these data strongly support the notion that intact signaling through an mGluR5-Homer2-PI3K complex within the NAC shell is important for a binge alcohol-drinking phenotype.
Figure 6 mGluR5-Homer binding is necessary for the “anti-binge” effects of NAC mGluR5 and PI3K blockade. Summary of the effects of an intra-NAC shell infusion of effective doses of MPEP and wortmannin upon the 5% alcohol intake in the SHAC procedure (more ...)
As blunted alcohol intake in Homer2
KO mice is associated with perturbations in glutamate receptor expression within the NAC (Szumlinski et al., 2004
), we next related the genotypic differences in binge drinking within the SHAC procedure to the basal expression of Homers, glutamate receptors and PI3K, as well as PI3K activity within the NAC. Consistent with the data for whole brain (), naïve mGluR5F1128R
mutants failed to exhibit significant differences from WT littermates in the total NAC protein expression of any of the glutamate receptor subtypes/subunits examined nor did they differ significantly in their total Homer1/2 protein expression (). While a moderate, but non-significant genotypic difference was observed regarding total PI3K expression, mGluR5F1128R
mutants exhibited an approximately 35% reduction in basal PI3K activity, as assessed by NAC levels of p(Tyr)p85α PI3K binding motif (t17
=2.56, p=0.02). These data confirm that the physical interaction between mGluR5 and Homers do not regulate their total protein expression (Tu et al., 1998
; ), but that this interaction is required for normal constitutive PI3K activity in vivo
. Moreover, these data provide our first evidence that a relationship exists between a genetic propensity for binge alcohol drinking under limited access conditions and the basal activational state of PI3K within the NAC.
Figure 7 mGluR5F1128R mutation reduces NAC basal PI3K activity. a, Representative immunoblots for the total protein levels of Homer2a/b, Homer1b/c, mGluR1, mGluR5, NR2a, NR2b, PI3K, p(Tyr)p85α PI3K binding motif [p(Try)p85α] and calnexin (loading (more ...)
Variance in NAC mGluR/Homer/PI3K expression between selectively bred SHAC and SLAC mice
To determine whether constitutive NAC PI3K activation represented a correlated response to selection, we next conducted immunoblotting on NAC tissue derived from 4th generation (S4) offspring of selectively bred male and female SHAC and SLAC mice. As is evident from an examination of the selection patterns for both alcohol intake and BACs (the selection phenotype), these responses vacillated across these early generations (), as would be expected of responses to selective breeding from an 8-way cross (see Crabbe et al., 2009 for discussion). The observation that the divergence in selection phenotype (BAC), but also alcohol intake, was asymmetrical and that parallel shifts in the absolute responses occurred between SHAC and SLAC lines across generations () suggests an environmental influence upon these measures (e.g., seasonal effects). Of relevance to this report, the selection procedures employed (see Methods for details) produced a significant divergence in limited access alcohol drinking across generations, as indexed by the alcohol intake of the mice () [Genotype effect: F(1,745) = 67.63, p < 0.001; Generation effect: F(3,745) = 19.44, p < 0.001; Interaction: F(3,745) = 2.61, p = 0.05] and the selection criterion of BACs attained following the 2nd 30 min alcohol drinking session in the SHAC paradigm () [Genotype effect: F(1,748)=68.46, p<0.001; Generation effect: F(3,748)=21.72, p<0.001; interaction: F(3,748)=5.44, p=0.001; n=80-104/line/generation]. The significant divergence in alcohol intake, as well as the selection phenotype of BAC, was evident by the 2nd generation of selection, and this divergence remained stable for the subsequent generations.
There was no significant line difference in water intake in offspring from generations S1, S3 and S4, although there was a slight, but significant increase in water consumption in the S2 SHAC vs. SLAC lines on days 2, 4 and 5 of testing (not shown). Importantly, there was no correlation between the water and alcohol intake exhibited by either group of mice during testing nor was there a line difference in total fluid intake on day 3 (alcohol + water) for any generation (i.e., SHAC mice increased their alcohol Intake relative to water, while SLAC mice increased their water intake relative to alcohol). Based on such data, in conjunction with the divergence in the lines in ethanol intake, it is unlikely that we were selecting simply on the basis of thirst. Unfortunately, as the feeding patterns of the mice were not measured during the first 3 hrs of the dark cycle, the possibility exists that our SHAC procedures may have selected for prandial food intake. However, our observation that body weight (and water intake) did not differ across days in the SHAC and SLAC lines, suggests also that we are not selecting for simple prandial factors. From the data depicted in , we estimated the heritability of the SHAC/SLAC trait from the multiple r2 from the ANOVA of the line difference in S4 BAC. Heritability was estimated as h2 = 0.16, indicating that 16% of individual differences in BAC after drinking were due to genetic influences. These data support the notion that binge alcohol drinking as assessed by the murine SHAC model is a genetically transmissible trait. Notably, the selection pressure and concomitant divergence of the selection phenotype alters the allele frequencies of genes that are relevant to the trait of interest, making selected lines a powerful tool to explore mechanisms underlying the selected trait.
As illustrated in , the S4 offspring of the SHAC and SLAC selected lines differed regarding NAC levels of Homer2a/b [F(1,12)=6.05, p=0.03], mGluR1 [F(1,12)=6.36, p=0.03], and p(Tyr)p85α [F(1,12)=10.43, p=0.008], with the SHAC line exhibiting greater protein expression in all cases, when compared to the SLAC line. While SHAC mice also exhibited elevated mGluR5 expression, this line difference was shy of statistical significance (p=0.09). These data are consistent with the data from the mGluR5F1128R mutant study (), as well as unpublished immunoblotting data from our laboratory derived from studies of inbred mice (Goulding, Obara, Lominac, Klugmann and Szumlinski, under review), and further the notion that genetic vulnerability to an excessive, binge alcohol drinking phenotype might relate to the basal functional status of the mGluR-Homer2-PI3K signaling pathway within the NAC.