Immunoblotting following alcohol drinking under DID procedures
The average alcohol intake exhibited by the B6 mice under DID procedures was approximately 4.8 g/kg/2 hour. As summarized in , DID drinking significantly increased NAC shell levels of Homer2a/b (t21=2.515, p=.02), mGluR5 (t22=3.806, p=.001), and NR2B (t22=2.129, p=.05). In contrast, the NAC shell levels of Homer1b/c, mGluR1, NR2A, and PI3K were unaffected by alcohol intake (all p’s > 0.10). Our data for both total and p-Ser473-Akt (the index of PI3K activity employed in this experiment) was highly variable and the DID-induced elevation in the relative amount of p-Ser473-Akt failed to reach statistical significance (p=.35). As shown in , with the exception increased Homer2a/b (t20=3.253, p=.004), no statistically significant alcohol-induced changes in protein expression were observed in the NAC core (all p’s>0.1). While we were unable to assay for core-shell distinctions in the relative expression p-Tyr(458)-PI3K due to insufficient tissue, an analysis of the relative levels of phospho-to-total levels of p85 within entire NAC tissue revealed an up-regulation of the phospho-to-total protein ratio (t21=2.16, p=.04), without significant alterations in total levels of either total p85 or p-Tyr(458)-PI3K (p’s>.25).
30 days of alcohol drinking under DID procedures up-regulated indices of glutamate signaling within the NAC
NAC blockade of mGluR5 and PI3K on alcohol drinking under DID procedures in B6 mice
An intra-NAC infusion of the mGluR5 antagonist MPEP reduced alcohol intake in the DID paradigm at the highest dose tested () [F(3,46)=3.05, p=0.04]. While the 3.0 µg MPEP dose reduced alcohol intake, it did not significantly affect water or 5% sucrose intake under identical conditions (; p>0.05). As reported previously under SHAC conditions (Cozzoli et al., 2009
), an intra-NAC infusion of the mGluR1 selective antagonist CPCCOEt (3 µg/side) produced a moderate, but non-significant reduction in alcohol consumption under DID procedures (VEH: 3.41 ± 0.66 g/kg; CPCCOEt: 2.18 ± 0.46 g/kg; p>0.05). An intra-NAC infusion of the non-selective PI3K inhibitor wortmannin (50 ng/side) reduced alcohol drinking by B6 mice and these effects were not additive with those of the mGluR5 antagonist MPEP () [F(4,38)=7.76, p<0.0001; post-hoc tests]. Similar to MPEP, an infusion of wortmannin failed to reduce significantly water or 5% sucrose consumption in these mice (; p>0.05), although the reduction in water intake approached statistical significance [t14
=1.973, p=.07]. Addressing the PI3K-specificity of the wortmannin effect, an infusion of highly PI3K-selective antagonist LY 294002 (0.17 ng/side) also reduced alcohol intake below that exhibited by animals infused with 0.1% DMSO vehicle, while infusion of the highly selective PLK-1 inhibitor cyclapolin 9 (38 pg/side) was ineffective () [Drug effect: F(2,12)=5.34, p=0.02; post-hoc tests].
Blockade of NAC shell mGluR5 and PI3K, but not mGluR1, reduces limited access alcohol drinking in B6 mice
Summary of the means ± SEM of the effects of intra-NAC infusion of vehicle, MPEP, and wortmannin on the intake of water and 5% sucrose (in ml’s) in the DID model.
mGluR5-Homer binding in binge alcohol drinking under DID procedures
Compared to their WT littermates, male F/R KI mice exhibited blunted alcohol drinking at both high (20%) and low (5%) alcohol concentrations () [Alcohol effect: F(1,14)=224.71, p<.0001; F/R KI effect: F(1,14)=5.39, p=.04; interaction: p=.22]. As illustrated in , the attenuating effects of our intra-NAC pharmacological manipulations upon the intake of 20% alcohol under DID procedures appeared to be selective for WT animals [Pretreatment × Genotype interaction: F(2,20)=7.389, p=0.004]. Deconstruction of our interaction along the Genotype factor confirmed in WT mice a significant reduction in drinking by both intra-NAC MPEP and wortmannin [F(2,10)=10.92, p=.003; post-hoc tests]. In contrast, neither of these manipulations altered drinking in KI animals (one-way ANOVA, p>0.05).
mGluR5-Homer binding is important for alcohol drinking under DID procedures and for the attenuating effects of NAC mGluR5 and PI3K blockade
Homer2 and binge alcohol drinking under DID procedures
In contrast to the results of our studies using the F/R KI mice, but consistent with earlier data for mGluR5 KO animals (e.g., Bledinov and Harris, 2008), Homer2 KO mice exhibited WT-levels of 20% alcohol intake under DID procedures when tested in the absence of any manipulation (). Despite this, WT-KO differences were observed regarding the attenuating effects of intra-NAC MPEP and wortmannin pretreatment upon the intake of 20% alcohol () [Pretreatment × Genotype: F(3,30)=3.93, p=.02]. Deconstruction of our interaction along the Genotype factor confirmed a significant reduction in drinking by intra-NAC MPEP and wortmannin in WT mice [F(3,15)=6.82, p=.004; post-hoc tests], but not in Homer2 KO animals (p>.05).
NAC shell Homer2 plays a role in mediating alcohol drinking under DID procedures and is necessary for the attenuating effects of NAC mGluR5 and PI3K blockade
To examine the possibility that some developmental compensation in the Homer2
KO mouse might be masking the effects of gene deletion upon limited access alcohol drinking, we investigated the impact of knocking-down Homer2b levels within the NAC shell upon alcohol consumption under DID procedures. Homer2b knock-down significantly reduced alcohol intake in the DID model and this effect was observed at both 5% and 20% alcohol, indicating a downward shift in the dose-response function by this pretreatment () [Alcohol effect: F(1,16)=388.69, p<.0001; shRNA effect: F(1,16)=6.94, p=.02; interaction: p=.14]. Moreover, this attenuation was specific to alcohol consumption as we showed previously in these same animals that NAC shell Homer2b knock-down did not affect water or sucrose intake (Cozzoli et al., 2009
Immunoblotting in selectively bred HDID-1 mice
As shown in , Homer2a/b (t26=−2.21, p=.036) and mGluR1 (t26=−3.289, p=.003) expression were increased significantly within the NAC shell in alcohol-naïve HDID-1 mice, compared to HS/Npt controls. While there appeared to be strain-dependent differences in the expression of NR2A, NR2B, total PI3K and phospho-p85α PI3K binding motif, these differences were not statistically reliable (for all proteins, p=0.09). Additionally, strain differences in protein expression seemed to occur preferentially within the NAC shell as no significant differences in protein expression were found in the NAC core ().
Differences in NAC shell Homer2 and mGluR1 expression in selectively bred high DID mice
Table 2 Comparison of the means ± SEM of the basal protein expression of Homer1b/c, Homer2a/b, mGluR1, mGluR5, NR2A, NR2B, PI3K, and p-(Tyr)p85α PI3K binding motif [p-PI3K] within the NAC core in the 15th generation offspring of mice selectively (more ...)