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The glutamate receptor-associated protein Homer2 regulates alcohol-induced neuroplasticity within the nucleus accumbens (NAC), but the precise intracellular signaling cascades involved are not known. This study examined the role for NAC mGluR-Homer2-PI3K signaling in regulating excessive alcohol consumption within the context of the Scheduled High Alcohol Consumption (SHAC) model of binge alcohol drinking. Repeated bouts of binge drinking (~1.5 g/kg/30 min) elevated NAC Homer2a/b expression and increased PI3K activity in this region. Virus-mediated knock-down of NAC Homer2b expression attenuated alcohol intake, as did an intra-NAC infusion of the mGluR5 antagonist MPEP (0.1-1 μg/side) and the PI3K antagonist wortmannin (50 ng/side), supporting necessary roles for mGluR5/Homer2/PI3K in binge alcohol drinking. Moreover, when compared to wild-type littermates, transgenic mice with an F1128R point mutation in mGluR5 that markedly reduces Homer binding exhibited a 50% reduction in binge alcohol drinking, which was related to reduced NAC basal PI3K activity. Consistent with the hypothesis that mGluR5-Homer-PI3K signaling may be a mechanism governing excessive alcohol intake, the “anti-binge” effects of MPEP and wortmannin were not additive, nor were they observed in the mGluR5F1128R transgenic mice. Finally, mice genetically selected for a high versus low SHAC phenotype differed in NAC mGluR, Homer2 and PI3K activity, consistent with the hypothesis that augmented NAC mGluR5-Homer2-PI3K signaling predisposes a high binge alcohol-drinking phenotype. Together, these data point to an important role for NAC mGluR5-Homer2-PI3K signaling in regulating binge-like alcohol consumption that has relevance for our understanding of the neurobiology of alcoholism and its pharmacotherapy.
Alcoholism is a chronic neuropsychiatric disorder affecting approximately 17.6 million people nation-wide (NIAAA, 2007). Alcohol act as a non-competitive antagonist at several glutamate receptors (e.g., Lovinger, 1996; Minami et al., 1998) and alters limbic glutamate neurotransmission, including that of the nucleus accumbens (NAC), which contributes to the motivation to drink and other properties of alcohol (c.f., Koob and Le Moal, 2008; Krystal et al., 2003; Siggins et al., 2003; DeWitte, 2004; Gass and Olive 2008). As observed following repeated alcohol injections (Szumlinski et al., 2005b, 2008b; Kapasova and Szumlinski 2008), binge-like alcohol consumption increases NAC extracellular glutamate levels and this response sensitizes with repeated bouts of binge-like drinking (Szumlinski et al., 2007). As NAC extracellular glutamate actively regulates alcohol intake (Kapasova and Szumlinski, 2008), molecular candidates contributing to the initiation and maintenance of excessive alcohol drinking are likely those regulating the development of alcohol-induced plasticity at glutamatergic synapses within the NAC.
The Homer2 member of the Homer family of postsynaptic scaffolding proteins has emerged as critical for NAC glutamate transmission and alcohol-induced neuroplasticity in vivo (c.f., Szumlinski et al., 2008a). Through an Ena/VASP1 Homology (EVH1) domain, Homers interact with a proline-rich motif (PxxF) located on Group 1 metabotropic glutamate receptors (mGluR1/5), as well as on other proteins involved in Group 1 mGluR intracellular signaling [incl. inositol-1,4,5-triphosphate (IP3) receptors, and the phosphatidylinositol-3 kinase enhancer-long (PIKE-L) complex] (e.g., Tu et al., 1998; Rong et al., 2003). A 3-month history of continuous alcohol intake up-regulates NAC Homer2 and mGluR1 protein expression in C57BL/6J (B6) mice (Szumlinski et al., 2008b) and virus-mediated NAC Homer2b over-expression augments various aspects of alcohol reward in mice, including free-access alcohol intake and alcohol-induced conditioned place-preference (Szumlinski et al. 2005b, 2008b). Conversely, Homer2 deletion in mice or D. Homer deletion in Drosophila results in an alcohol-avoiding phenotype (Szumlinski et al., 2005b; Urizar et al., 2007), which resembles the “anti-alcohol” effects produced by systemic or intra-NAC administration of mGluR1/5 antagonists (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; 2008b; Blednov and Harris, 2008). These preclinical data, coupled with reports of associations between single nucleotide polymorphisms (SNPs) in genes encoding glutamate receptors or the p85 regulatory subunit of PI3K with risky alcohol drinking behavior in adolescents (Desrivieres et al., 2008; Schumann et al., 2008) have led to the hypothesis that Group 1 mGluR-Homer2-PI3K signaling within the NAC is necessary for stable, excessive alcohol consumption. Thus, we employed a combination of proteonomic and in vivo pharmacological and genetic approaches to characterize the role for this signaling cascade in regulating sustained, excessive alcohol consumption using the murine SHAC (Scheduled High Alcohol Consumption) model (Finn et al., 2005), one of several rodent models of binge alcohol drinking (for review, see Rhodes et al, 2005). The results of this study provide novel evidence that binge alcohol drinking-induced increases in NAC mGluR5-Homer2-PI3K pathway activation is necessary for this prevalent form of alcohol-directed behavior and that basal NAC PI3K activity may contribute to genetic variance in binge alcohol drinking behavior.
The majority of subjects employed in this study were adult male, inbred C57BL/6J (B6) mice (8 weeks of age; 25-30 g; Jackson Laboratories, Bar Harbor, ME), that were allowed to acclimate to the colony room for at least 7 days following arrival. For all drinking experiments, animals were single-housed in polyethylene cages in a temperature (25 ° C) and humidity (71%) controlled colony room under a 12-h reverse light cycle (lights off at 0500 h; lights on at 1700 h), otherwise mice were housed in groups of 4. Food was available ad libitum and water access was restricted in the drinking studies as described below. All experimental protocols were approved by the IACUC of our respective institutions and were consistent with the guidelines provided by the National Institute of Health (NIH) Guide for Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996).
To examine the functional relevance of mGluR5-Homer interactions for binge drinking behavior and protein expression, a transgenic mouse with a phenylalanine (F)→arginine (R) point mutation at amino acid position 1128 of mGluR5 was generated using the strategy presented in Figure 1a. The mGluR5F1128R targeting construct was generated based on a 13kb EcoRV fragment from a DNA BAC clone including mouse mGluR5 gene exon 8. Selective markers were introduced by inserting a lox-p flanked PGK-Neo fragment (including Sph I and Xba I restriction sites) in the unique Pac I site of exon 8. F1128R mutations were introduced by replacing a 1.2kb DNA fragment with two point mutations (TTC to CGC) downstream of the Pac I site in exon 8 (0.3kb belonging to intron 7 and 0.9kb belonging to the exon 8 coding region) (see Figure 1c). Thus, the construct has a 9.8kb long arm and 2kb short arm for homologous recombination. The resulting targeting construct was linearized and electroporated into R1 ES cells. Cells were selected with G-418 for two weeks. Clones were picked, screened by PCR, and confirmed by Southern blotting (Figure 1b). Positive clones were injected into blastocysts, and chimeras were mated to C57BL/6J mice to produce mGluR5F1128R heterozygotes. PGK-Neo was removed by crossing these heterozygotes with actin promoter-driven cre mice. To verify that the F1128R mutation reduced the physical interaction between Homers and mGluR5, co-immunoprecipitation was conducted on brain tissue from wild-type (WT) and mGluR5F1128R TG mice. Whole brains were dissected and sonicated in an immunoprecipitation (IP) buffer (1 × PBS, pH 7.4, with 5 mM EDTA, 5 mM EGTA, 1 mM Na3VO4, 10 mM sodium pyrophosphate, 50 mM NaF, and 1% Triton X-100) containing Complete™ EDTA-Free protease inhibitors. Samples were centrifuged at 13,000 rpm for 30 min at 4°C. The supernatant (300 ml) was then mixed with 0.5-2 μg of the appropriate antibody for 3 hours at 4°C. Then 50 ml of 1:1 protein A- or protein G-Sepharose slurry (Amersham-Pharmacia Biotech; Picscataway, NJ) was added for an additional 1 h. The protein beads were washed three times with IP buffer containing 1% Triton X-100. The protein samples were separated electrophoretically using NuPAGE 4-12% Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred to an Immobilon-P PVDF membrane (Millipore; Billirica, MA). The membrane was blocked with TBST (50 mM Tris, pH 7.5, with 150 mM NaCl, and 0.1% Tween-20) containing 5% non-fat milk for 1 hour at room temperature, followed by incubation with primary antibody in TBST buffer overnight at 4°C. After 3 washes with TBST buffer, membranes were incubated with HRP-conjugated anti-rabbit antibody in TBST for another hour. After 3 washes with TBST buffer, the membrane was treated with SuperSignal ECL substrate (Pierce) according to the manufacturer's protocol. Using this procedure, immunoblotting verified that the point mutation interfered with the capacity of Homers to bind to mGluR5 (Figure 1e), without affecting total mGluR5 expression (Figure 1d) (student's t-test, p=0.13, n=4). A follow-up examination of accumbens tissue also confirmed that the point mutation did not significantly affect total mGluR5 expression, nor did it alter the expression level of either Homer1b/c or Homer2a/b protein (see Figure 8).
For the present experiments, male wild-type (WT) and homozygous mGluR5F1128R transgenic (TG) littermate mice were generated from heterozygous breeder pairs (F10-12; C57BL/6J × 129Xi/SvJ), housed under conditions described for the B6 mice above. All testing for behavior or for changes in protein expression commence when mice were 7-8 weeks of age.
“Binge” alcohol drinking is defined by NIH as alcohol consumption that brings blood alcohol concentrations (BACs) to 0.8 mg/ml within an approximately 2 hr-drinking period (NIAAA, 2004) and is the most prevalent pattern of heavy alcohol consumption exhibited within the United States (NIAAA, 2007). Given this, we chose to conduct selective breeding to optimize the genetic contribution to the “binge drinking” phenotype (see subsection below) and produce lines of mice that differed in their limited-access alcohol intake. One advantage of selected lines is that they can be used to identify genetically correlated traits. That is, the appearance of a significant difference in the selection phenotype, as well as in another trait, implies the existence of pleiotropically acting genes. Additionally, the selection pressure 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. Many of the characteristics of selected lines for use in evaluating genetic correlation, as well as the theoretical considerations involved in their construction and evaluation of practical/theoretical tradeoffs, have been discussed previously (Crabbe et al., 1990; Crabbe, 1999). To examine whether or not the NAC expression of glutamate receptors, Homers and PI3K activity were genetically correlated with a binge drinking phenotype, genetically heterogeneous mice (HS/Npt), created by Dr. Robert Hitzemann and maintained at Oregon Health & Science University, served as the founding population for the generation of Scheduled High and Low Alcohol Consumption (SHAC/SLAC) selected lines. The selection of the SHAC and SLAC selected lines began with testing 121 HS/Npt mice (59 male and 62 female) on the selection version of the SHAC procedure. Briefly, following acclimation to individual housing, mice had access to 4 hr fluid/day. On days 1 & 2 and days 4 & 5, mice had access to tap water for 4 hr. On day 3, mice initially had access to a 5% alcohol solution for 30 minutes, followed by access to tap water for 3.5 hrs. As binge alcohol drinking is defined in terms of BACs attained following limited access drinking (NIAAA, 2007), on day 6, a retro-orbital blood sample was taken to assess BAC immediately after the 30-min alcohol session (see Finn et al., 2007). The behavioral experiment was terminated at this point, and animals were maintained on ad libitum food and water until decisions on breeders were made (i.e., after all animals had been tested). It was decided that the selection phenotype for the SHAC and SLAC lines would be based on the BAC on day 6, since pilot studies determined that BAC and alcohol dose consumed on day 6 were positively correlated with these dependent variables on day 21 (when animals had 10 hrs of total fluid availability), indicating that the alcohol intake patterns on day 6 were representative of intake patterns when animals were no longer fluid restricted.
The starting stock of HS mice that was tested initially (S0) contained representative animals from 44 of the 45 families that comprised the HS/Npt mice. The alcohol intake and BAC on day 6 in all animals was 1.50 g/kg and 1.05 mg/ml, respectively. As we had proposed to start with mass selection, we chose 12 primary and 3 alternate breeding pairs to begin propagating the SHAC line, representing 20 families. The animals chosen were those with the highest BACs. Alcohol intake and BAC was 1.97 g/kg and 1.59 mg/ml in the breeders for the SHAC line. Likewise, we chose 12 primary and 3 alternate breeding pairs (mice with the lowest BACs) to begin propagating the SLAC line, representing 22 families. Alcohol intake and BAC was 0.924 g/kg and 0.531 mg/ml in the breeders for the SLAC line. After choices for S1 breeders were determined, the breeding strategy was switched to within family selection. A within family selection breeding strategy reduces the rate of inbreeding to approximately one-half of that seen with mass selection by systematically reducing the (already small) degree of genetic relatedness of each pair of mice mated to produce the next generation (see Falconer and Mackay, 1996; Crabbe, 1999 for detailed discussions). Beginning with testing of the S1 offspring, choices for S2 breeders were made by picking one male and female from each family of the SHAC line (with the highest BAC on day 6) for the SHAC breeders and one male and female animal from each family of the SLAC line (with the lowest BAC on day 6) for the SLAC breeders. A similar strategy was used to produce each subsequent generation. The SHAC and SLAC selected lines were maintained in reproductive isolation from each other starting with S1. A rotational breeding scheme was used so that mating pairs were constructed to exclude littermates, as well as common grandparents, whenever possible. The proportion of animals selected to serve as breeders from the S0 generation was 25%, and the breeder proportion did not exceed 16% in subsequent generations (i.e., 15 breeding pairs for each line, testing approximately 90-100 offspring per line). Additional consideration was given to the relationship between BAC and alcohol dose on day 6 (to ensure that high intake corresponded to high BAC and visa versa), to the relationship between alcohol dose on days 3 and 6 (to ensure that consumption had not dropped on day 6), as well as to significant (± 20%) changes in body weight. There was never a circumstance where we had to substitute another breeder for our first choices of SHAC and SLAC mating pairs, based on these additional considerations. Scheduled water consumption was not considered as a potential confounding variable, as we had previously observed that water consumption under a similar fluid restriction schedule did not differ markedly between animals on the SHAC procedure or similarly treated water controls (Cronise et al., 2005).
Due to financial limitations, the selection study was terminated after testing of the S4 offspring was completed. Three months after the offspring were tested for intake, the S4 SHAC and SLAC mice were killed. Whole brain was removed from a subset of the S4 offspring from each selected line and shipped frozen to the University of California at Santa Barbara for subsequent immunoblotting procedures. As described in the Results and depicted in Figure 9b, the significant divergence in BAC in the SHAC and SLAC lines beginning in the S2 generation provides support for the use of these lines as a genetic animal models of “binge drinking” and “low binge drinking”, respectively.
One of the major criticisms of the majority of existing rodent models of alcohol drinking is that rarely do the animals achieve pharmacologically relevant blood alcohol concentrations during alcohol access (i.e. BAC > 0.8 mg/ml) nor do they maintain pharmacologically significant BACs over extended periods of alcohol access (e.g., Dole and Gentry, 1984; Le et al., 2001; Graham and Grose, 2003). While earlier data (Szumlinski et al., 2005b, 2008b) indicated an important role for NAC Homer2 function in moderate alcohol intake under both operant and non-operant alcohol self-administration procedures, the role for Group1 mGluRs and the intracellular mediators of mGluR5/Homer2 activation in maintaining excessive, binge-like alcohol drinking is not known. To address this issue, the present study employed a variation of the murine SHAC model of excessive, binge-like alcohol drinking, originally described by Finn et al. (2005). The SHAC procedures employed were identical to those described recently in Szumlinski et al. (2007) and involved the presentation of a single 50 ml sipper tube containing either water or a 5% (v/v) alcohol solution for 30-min, starting at 3 hours into the circadian dark cycle. Alcohol presentation occurred every 3 days, with water presentation on intervening days. Following each 30-min drinking session, the home cage water bottle was returned and mice were allowed to consume water for an additional 9.5 hours (total fluid availability/day = 10 hrs). At this time (1800 h), the home cage water bottle was removed. The amount of water and/or alcohol consumed during the 30-min session was determined daily by bottle weight prior to, and following, each 30-min drinking session and alcohol/water intake was calculated respectively on a g/kg and ml/kg body weight basis. As mice consume the majority of their daily fluid intake during the circadian dark period (e.g., Middaugh et al., 1999), our modified SHAC procedures involved fluid restriction throughout the circadian light period into the first 3 hrs of the circadian dark phase (Szumlinski et al., 2007). Despite eliciting high levels of alcohol intake (1.3 – 1.9 g/kg/30 min) and correspondingly high BACs (>1.0 mg/ml; Szumlinski et al., 2007), this amount of fluid restriction is considered minimal as it does not promote excessive water intake upon a 30-min presentation of a sipper tube containing water as assessed earlier in pilot experiments (non-water-restricted: 9.9 ± 3.62 ml/kg vs. water-restricted: 15.41 ± 6.05 ml/kg, n=11, t10=1.12, p>0.05). Our data for water intake in this extended SHAC procedure are consistent with earlier SHAC studies employing longer periods of fluid deprivation (Cronise et al., 2005; Finn et al., 2005), as well as other data (Toth and Gardiner, 2000) indicating that fluid-restricted mice adapt and do not exhibit a compensatory increase in fluid consumption or other detrimental effects, such as a reduction in feeding or weight loss. As binge alcohol drinking is defined by a BAC > 0.8 mg/ml within a 2-hr period (NIAAA, 2007), we conducted a regression analysis on the relationship between alcohol intake during the 7th 30-min drinking session and the BACs attained by male B6 mice (n=45; Figure 2). As reported previously (Finn et al., 2005), this analysis indicated a relationship between these two variables that was significantly different from 0 [F(1,43)=26.39, p<0.0001] and the resulting equation, BAC = 0.28 * (intake) + 0.59, was employed to determine whether or not our transgenic and pharmacological manipulations, by definition, prevented binge drinking behavior in our mice. Additionally, the goodness of fit (r2) of the regression line was 0.38, indicating that 38% of the variability in BEC could be accounted for by the variation in alcohol dose consumed.
Chronic, continuous alcohol consumption elevates NAC levels of Homer2, and associated glutamate receptor proteins (Szumlinski et al., 2008b). To determine whether or not alcohol drinking up-regulates the mesocorticolimbic expression of members of the mGluR-Homer-PI3K signaling pathway in the fully extended SHAC drinking model (for discussion, Finn et al., 2005), B6 mice were subjected to 6 bouts of SHAC drinking over an 18-day period with 5% alcohol available for 30 min, every 3rd day (see above). Control animals received tap water in an identical 50 ml sipper tube during each of the 30-min sessions. Animals were decapitated 24 hrs following the 6th alcohol presentation, brains were sectioned (1.0 mm thick) along the coronal plane and the entire prefrontal cortex, NAC, dorsal striatum and hippocampus were dissected out over ice. As the mGluR5F1128R mutation reduced binge drinking with the SHAC procedure (see Results), a second experiment assessed genotypic differences in basal NAC protein expression in experimentally naïve WT and mGluR5F1126R mutant mice. Finally, a 3rd immunoblotting experiment was conducted on NAC tissue from selectively bred SHAC and SLAC mice (see above) to further relate genetic vulnerability in binge alcohol drinking to mGluR/Homer/PI3K expression in the NAC (i.e., was mGluR5/Homer/PI3K expression in the NAC a correlated response to selection for binge drinking?). For this experiment, frozen whole brains from S4 SHAC and SLAC offspring 3 months following alcohol testing were sectioned along the coronal plane (1 mm thick) at the level of the NAC and the entire NAC and dorsal striatum dissected out over ice.
As described in recent reports by our group (Ary and Szumlinski, 2007; Ary et al., 2007; Szumlinski et al., 2008b), the tissue from all the experiments outlined above was homogenized in a medium consisting of 0.32 M sucrose, 2 mM EDTA, 1% w/v sodium dodecyl sulfate, 50 μM phenyl methyl sulfonyl fluoride and 1 μg/ml leupeptin (pH=7.2) and 50 mM sodium fluoride, 50 mM sodium pyrophosphate, 20 mM 2-glycerol phosphate, 1 mM p-nitrophenyl phosphate, and 2 μM microcystin LR were included to inhibit phosphatases. Samples were then subjected to low-speed centrifugation at 10,000 g for 20 min. Protein determinations were performed using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA), according to the manufacturer's instructions and homogenates were stored at -80°C until immunoblotting was completed.
For immunoblotting, protein samples (5-20 μg) were subjected to a SDS-polyacrylamide gel electrophoresis. Bis-Tris gradient gels (4-12%) (Invitrogen) were used for separation of Homers, PI3K, and the p(Tyr)p85α PI3K binding motif, the latter of which was employed to index PI3K activity (e.g., Zhang et al., 2006). Tris-Acetate gradient gels (3-8%) (Invitrogen) were used for separation of Homers, as well as the glutamate receptor proteins. Proteins were transferred to PVDF membranes, preblocked with phosphate-buffered saline containing 0.1% (v/v) Tween-20 and either 5% (w/v) bovine serum albumin [for p(Tyr)p85α PI3K binding motif] or 5% (w/v) nonfat dried milk powder (for all other proteins) for no less than 1 hr before overnight incubation with primary antibodies. The following rabbit polyclonal antibodies were used: anti-Homer 2a/b and anti-Homer 1b/c (Dr. Paul F Worley, Johns Hopkins University School of Medicine; 1:1000 dilution), anti-mGluR5 (Upstate Cell Signaling Solutions, Lake Placid, NY; 1:1000 dilution), anti-NR2a and anti-NR2b (Calbiochem, San Diego, CA; 1:1000 dilution), anti-PI3K antibody (Upstate, Lake Placid, NY; 1:1000 dilution), and anti-p-(Tyr) PI3K p85α binding motif (Cell Signaling Technology, Beverly, MA; 1:500 dilution). An anti-mGluR1a mouse polyclonal antibody (Upstate, Lake Placid, NY; 1:1000 dilution) was also used. Membranes were washed, incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary anti-body (Upstate, Charlottesville, VA; 1:20,000-1:40,000 dilution) or anti-mouse secondary anti-body (Jackson Immuno Research Laboratories, West Grove, PA; 1:20,000-1:40,000) for 90 min, washed again, and immunoreactive bands were detected by enhanced chemiluminescence using either ECL Plus (Amersham Biosciences) or Pierce SuperSignal West Femto (Thermo Fisher Scientific, Rockford, IL). A rabbit anti-calnexin polyclonal primary antibody (Stressgen, Victoria, BC) was also used to index protein loading and transfer. The levels of immunoreactivity for all proteins were quantified using Image J (NIH, Betheseda, MD) and the immunoreactivity for each protein of interest for each animal was first normalized to that of its appropriate calnexin signal to provide a protein/calnexin ratio. These ratios were then normalized to the mean ratios for each protein of the water or genetic control for each individual gel (n=3-4/gel).
The surgical procedures for implanting bilateral guide cannulae into the NAC of mice (i.e., B6, WT or TG) were similar to those employed in previous studies (e.g., Lominac et al., 2006; Szumlinski et al., 2007, 2008b; Kapasova and Szumlinski, 2008). Mice were anesthetized by inhalation of isoflurane with 4% oxygen as the carrier gas. Mice were mounted in a Kopf stereotaxic device with tooth and ear bars adapted for mice. The animal's skull was exposed, leveled, and holes were drilled based on a set of coordinates from Bregma for the NAC shell (AP: +1.3; ML: ±0.5 mm; DV: -2.3 mm) or NAC core (AP: +1.3; ML: ±0.7 mm; DV: -2.0 mm), according to the mouse brain atlas of Paxinos and Franklin (2004). The skull was prepared for polymer resin application and stainless steel guide cannulae (20-gauge, 10 mm long; Small Parts, Roanoke, VA) were lowered such that the tips of the cannulae were 2 mm above the NAC. A mound of resin was placed around the guide cannulae for stabilization and then light cured. The incision was closed with a tissue adhesive. To prevent continuous externalization, dummy cannulae (24-gauge; length equivalent to guide cannula) were placed inside the guide cannulae and only removed prior to drug testing. Following surgery, the animals are allowed to recover for 4-5 days prior to the start of the SHAC drinking procedures.
Our earlier studies conducted in B6 and B6-hybrid mice demonstrated a facilitatory role for NAC Homer2 expression in alcohol intake, under both non-operant and operant conditions (Szumlinski et al., 2005b, 2008b). As repeated bouts of binge drinking were found to elevate NAC expression of Homer2 (see Results), we employed a similar adeno-associated viral vector (AAV) gene-transfer approach to examine the functional relevance of increasing and decreasing NAC Homer2 expression for binge drinking behavior. The procedures for generating AAVs carrying Homer2b cDNA have been described in detail in Klugmann et al. (2005) and Klugmann and Szumlinski (2008). In brief, Homer2b was expressed as an N-terminal fusion protein with the hemagglutinin (HA)-tag in a recombinant AAV backbone containing the 1.1 kb CMV immediate early enhancer/chicken β-actin (CBA) promoter (AAV-Homer2b). The same backbone encoding renilla green fluorescent protein (hrGFP) was used as control (AAV-GFP). For generation of Homer2b-specific small hairpin RNAs (shRNAs), we searched the Homer2b mRNA using a shRNA Design Algorithm (Ambion) and identified the targets sequences H2b#1 (5′-GUGUGAAUAUGUCUCUGAGTT-3′) and H2b#2 (5′-CACAGAGUGCUGCCAAUGUTT-3′). Similarly, a sequence (5′-ACUACCGUUGUUAUAGGTGTT-3′) with no significant homology to any endogenous target (Universal negative control, UNC) was cloned to serve as a negative control for potential non-specific effects mediated by delivery of shRNA. We generated sense and antisense oligonucleotides corresponding to these targets, annealed them and cloned them into an AAV plasmid that simultaneously drives the expression of the shRNAs and hrGFP as previously described (Franich et al., 2008). In this cassette, shRNAs for knockdown of Homer2b were driven by the human U6 promoter inserted upstream of the CBA promoter in the AAV-GFP vector (Franich et al., 2008). This bicistronic strategy allowed for easy identification of cells transduced by the shRNA expressing vector. Packaging of chimeric AAV1/2 vector was performed as described (Klugmann et al., 2005) and genomic titers were determined using a Prism 7700 sequence detector system (Applied Biosystems, Foster City, CA) with primers designed to WPRE (During et al., 2003).
The procedures for infusing AAVs were identical to those employed in earlier mouse studies (Szumlinski et al., 2004, 2005b, 2008b; Lominac et al., 2005). One week following surgery, 33-gauge injector cannulae (12 mm long; threaded through a 24-gauge adapter for stability) were lowered bilaterally into the NAC and AAVs infused at a rate of 0.1 μl/min for 5 min (total vol=0.50 μl/side). A similar infusion procedure produces neuronal transduction that is restricted to less than 1 mm of the infusion site and is maximal for both AAV-cDNAs and AAV-shRNAs at 3 weeks post-infusion (Szumlinski et al., 2004, 2005b, 2006, 2008b; Lominac et al., 2005; Klugmann and Szumlinski, 2008; see also Figure 4). In the case of the shRNAs, AAV infusion of 0.5 μl/side produces a 50% reduction in total Homer2 protein expression within the NAC, without affecting Homer1b/c protein expression, as assessed by immunoblotting (Klugmann and Szumlinski, 2008). For all experiments involving intra-NAC AAV infusion, testing began 3 weeks later. As we examined the site-specificity of Homer2b manipulations upon binge drinking behavior, following AAV testing, mice were perfused transcardially with saline, followed by a 4% paraformaldehyde solution and immunocytochemical staining for the HA tag was performed to localize the extent of transduction efficiency within the NAC and to examine for gross signs of neurotoxicity, as conducted previously (Szumlinski et al., 2004, 2005b, 2006, 2008b; Klugmann et al., 2005; Lominac et al., 2005). As described in greater detail below, in all, 3 distinct AAV studies were conducted in series (respectively, NAC shell cDNA, NAC core cDNA and NAC shell shRNA) with 3-4 months intervening between studies. For each study, mice were tested in 2 cohorts of 5-6 mice/AAV treatment/cohort and the cohorts were spaced 2 months apart.
To establish a necessary role for NAC Group1 mGluR and PI3K activation in the maintenance of excessive alcohol drinking, the effects of the local infusion of mGluR5, mGluR1 and PI3K antagonists upon “binge drinking” with the SHAC procedure were determined in 3 separate experiments (1 per compound). In these behavioral pharmacological experiments, mice were presented with 5% alcohol for 30 min, every 3rd day, until stable intake was established (less than 10% variability across 3 consecutive presentations; ~3-4 presentations). MPEP [2-methyl-6-(phenylethynyl)pyridine hydrochloride; 0, 0.1, 0.3, and 1.0 μg/side; Sigma-Alrich Chemical Co., Natick, MA], the mGluR1a antagonist CPCCOEt [7-(hydroxyimino)cyclopropa[b] chromen-1a-carboxylate ethyl ester; 0, 1.0, and 3.0 μg/side; Tocris Cookson, Ellisville, MO], and a PI3K-selective dose of the antagonist wortmannin (0 vs. 50 ng/side; Sigma-Alrich; Rong et al., 2003) were infused through a 33-gauge injector (12 mm long; threaded through a 24-gauge adapter for stability) at a rate of 0.25 μl/min for a total volume of 0.25 μl/side using a Harvard PhD2000 automated syringe pump and the injectors remained in place for an additional 60 sec. MPEP and wortmannin were dissolved in sterile water for infusion and CPCCOEt was dissolved in water containing cylcodextrin (45% w/v; Sigma-Alrich) and water or a 45% w/v cyclodextrin solution served as the appropriate control (e.g., Schroeder et al., 2005; Hodge et al., 2006; Besheer et al., 2008a; Lominac et al., 2006). In these antagonist studies, an intra-NAC infusion of neither the water nor cyclodextrin vehicle affected alcohol intake relative to that exhibited by these same mice in the absence of any microinjection (e.g., non-infused: 1.31 ± 0.12 g/kg vs. water-infused: 1.44 ± 0.09 g/kg vs. cyclodextrin-infused: 1.42 ± 0.09 g/kg), indicating that if any disruption in the osmotic balance of the NAC was produced by our vehicle pretreatments, it did not influence binge alcohol drinking. Immediately following infusion, mice were returned to their home cage and presented with the 5% alcohol-containing sipper tube for 30 min. Within each experiment, mice received at least 2 alcohol bottle presentations between intra-NAC drug tests to examine for potential carry-over effects of pretreatment and to ensure that alcohol drinking had stabilized. In none of the experiments were carry-over effects observed upon either alcohol or water intake on the intervening days (i.e., there were no significant differences between the average pre-test intake and intake on the intervening non-test days; data not shown). The order of the dosing was randomized across test days and, to reduce the amount of tissue damage caused by repeated testing and to reduce the risk of infection, mice were tested with a maximum of 3 doses (i.e., not all mice in the MPEP study received all antagonist doses). To avoid disruption of drinking behavior on subsequent test days, BACs were not assessed but rather were predicted based on the mean alcohol intake according to the results of the regression analysis provided in Figure 2. To assess for non-specific effects of intra-NAC antagonist infusion, the maximally effective dose for reducing alcohol intake was then examined upon water intake during a 30-min session. Standard cresyl violet histochemical procedures were employed to verify injector cannulae localization in the NAC shell (e.g., Szumlinski et al., 2005b, 2008b; Lominac et al., 2006; Kapasova and Szumlinski, 2008).
NAC Homer2 over-expression and intra-NAC infusion of a glutamate reuptake inhibitor increase, while Homer2 or mGluR5 deletion and systemic pretreatment with mGluR5 antagonists reduce, alcohol intake of B6 mice under continuous alcohol access procedures (Backstrom et al., 2004; Olive et al., 2005; Szumlinski et al., 2005b; Lominac et al., 2006; Besheer et al., 2008a; Blednov and Harris, 2008; Szumlinski et al., 2008b). Thus, we next determined whether or not the effects of the mGluR5F1128R mutation upon drinking within the SHAC procedure (see Results) generalized to voluntary alcohol preference drinking. For this, an independent study examined the mGluR5F1128R TG and WT mice for genotypic differences in alcohol intake and preference using a 4-bottle-choice procedure as previously described (Backstrom and Hyytia, 2004; Szumlinski et al., 2005b, 2008b; Lominac et al., 2006). In brief, WT and TG mice were presented simultaneously with four 50 ml sipper tubes containing 0, 3, 6 and 12% alcohol (v/v) and allowed to drink undisturbed in the home cage over a 24-hr period. For 3 weeks, bottles were weighed daily at 10:00 h and the total alcohol intake was calculated on a g/kg weight basis. The preference for each alcohol concentration was calculated as the percent of the total fluid consumed from the 4 bottles. The average alcohol and water intake, as well as alcohol preference, during the last week of testing were employed in the analysis of genotypic differences. In both the SHAC and continuous alcohol access procedures, spillage due to bottle manipulation was monitored by weighing the appropriate number of bottles on empty cages and the average spillage for a given day (0.05-0.1 ml) was subtracted from the experimental data for that day.
Immunoblotting data were statistically evaluated using Student's t-tests. The behavioral data for the AAV-cDNA studies were analyzed using unpaired Students t-tests (Control vs. cDNA) and that for the AAV-shRNA study were analyzed using a between-subjects analysis of variance with 3 levels on the AAV factor (Control, shRNA1 and shRNA2). The attrition rate in the AAV studies was 1-2 animals/group due to caudally misplaced microinjectors (i.e., in ventral pallidum). As not all mice received all intracranial treatments either due to experimental design (maximum of 3 microinjections) or loss of guide cannulae patency (maximum 2 mice/experiment), to include all of the animals with viable data, the alcohol intake data for the MPEP, wortmannin and CPCCOEt studies were analyzed using a between-subjects ANOVA with 4 levels on the MPEP dose factor, 3 levels on the wortmannin dose factor and 3 levels on the CPCCOEt dose factor, respectively. As there was no attrition in the study examining the effects of either antagonist infusion upon water intake, these data were analyzed using paired t-tests (control-infused vs. drug-infused). The data for mGluR5F1128R mutant studies were analyzed using between-subjects (WT vs. TG) univariate ANOVAs, with the exception of the data for alcohol preference, which was analyzed using a mixed-design ANOVA (Genotype × Alcohol), with 4 levels on the within-subjects factor of Alcohol (0, 3, 6, and 12% alcohol). There was no attrition due to loss of guide cannulae patency or misplaced microinjector placements in the studies examining the combined effects of intra-NAC drug infusion and the mGluR5F1128R mutation upon behavior, thus, the data were analyzed using a mixed-design ANOVA (Genotype × Drug), with 3 levels on the within-subjects factor of Drug (vehicle, MPEP, wortmannin). When appropriate, post-hoc comparisons were made using Fischer's least significant difference test.
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; Figure 2) 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 Figure 3 and Table 1, 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 (Figure 3). 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 (Figure 3). 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.
In contrast to the data for the NAC (Figure 3), 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 (Table 1). However, as summarized in Table 1, 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.
Homer2 expression within the NAC shell actively regulates alcohol intake under continuous access and operant self-administration conditions (Szumlinski et al., 2005b, 2008b). As the SHAC procedure elicited a large rise in the NAC total protein expression of Homer2a/b (Figure 3), 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, 2005b, 2006, 2008b; 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 Figure 4a and its subpanels (target = NAC core), transfection was observed within both cell bodies and processes within 1 mm of the infusion site (Figure 4a″).
In contrast to the data obtained using other animal models of alcohol reward/intake (Szumlinski et al., 2005b, 2008b), 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 (Figure 4b). As illustrated in Figure 4c, 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; 2005a; 2005b; 2008b). 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 (Figure 4b) or water intake (Figure 4c). 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% (Figure 4b) [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 (Figure 2), 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, 2005a, 2005b), NAC shell Homer2b knock-down did not affect the average water intake of the mice exhibited during testing (Figure 4c), 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.
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, 2008b; 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 (Figure 3), 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; Figure 5a) 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.
As illustrated in Figure 5a, 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 [Figure 5a; 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 (Figure 5a). 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 (Figure 4c) 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 (Figure 5b). 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.
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 (Figures 4 and and5;5; 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.
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 (Figure 6) [Genotype effect: F(1,11)=4.32, p=0.05; Drug effect: F(2,22)=4.50, p=0.02]. The data in Figure 6 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.
As blunted alcohol intake in Homer2 KO mice is associated with perturbations in glutamate receptor expression within the NAC (Szumlinski et al., 2004, 2005), 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 (Figure 1d, 1e), 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 (Figure 7). 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; Figure 1d, 1e), 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.
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 (Figure 8), 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 (Figure 8a and 8b) 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 (Figure 8a) [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 (Figure 8b) [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 Figure 8b, 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 Figures 8c and 8d, 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 (Figure 7), 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.
The present report provides in vivo validation of the involvement of an mGluR5-Homer2-PI3K signaling pathway within the NAC shell in binge alcohol-drinking behavior by showing that the site-directed pharmacological and transgenic interruption of this pathway reduces the extent to which mice drink alcohol within the context of the SHAC model of alcoholism. This finding extends earlier indications of important roles for Homer2 and mGluR5 in regulating alcohol reward in various rodent models of alcohol intake (e.g., Backström et al., 2004; Olive et al., 2005; Schroeder et al., 2005; Szumlinski et al., 2005b, 2008b), as well as an association between polymorphisms in genes encoding glutamate receptors, as well as SNPs in the p85 regulatory subunit of PI3K with risky alcohol drinking behavior in human adolescents (Desrivieres et al., 2008; Schumann et al., 2008). As observed following chronic, moderate alcohol intake (Szumlinski et al., 2008b), repeated bouts of binge drinking elevate NAC Homer2 levels, and consistent with in vitro evidence that Homer proteins complex with the PI3K-enhancer molecule PIKE-L to regulate constitutive PI3K activity (Rong et al., 2003), the binge drinking-induced rise in NAC Homer2 was concomitant with increased basal PI3K activation. Moreover, an examination of both mutant and selectively bred mouse lines revealed a relationship between genotypic differences in binge alcohol drinking and basal NAC expression of Homer2, Group1 mGluRs and/or PI3K activity. Taken altogether, these results implicate idiopathic and alcohol drinking-induced increases in the functional status of the mGluR5-Homer2-PI3K signaling pathway within the NAC shell subregion in regulating the propensity to binge drink alcohol under limited access conditions.
Clinical and preclinical data support a relation between the propensity to consume/prefer large amounts of alcohol, a reduction in behavioral sensitivity to alcohol, and alcohol-induced increases in metabolic activity or glutamate release within the NAC or within its major glutamatergic afferent structures (Selim and Bradberry, 1996; McBride et al., 1986; Dahchour et al., 2000; Moselhy et al., 2001; Piepponen et al., 2002; Meyerhoff et al., 2004; Kapasova and Szumlinski, 2008). Repeated bouts of binge alcohol drinking under SHAC procedures sensitizes the capacity of ingested alcohol to elevate NAC levels of extracellular glutamate (Szumlinski et al., 2007) and pharmacological manipulations of NAC glutamate actively regulate alcohol ingestion in mice (Kapasova and Szumlinski, 2008).
Alcohol inhibits mGluR5 function (Minami et al., 1998) and the therapeutic efficacy of acamprosate for treating alcoholism is hypothesized to relate to a reduction in alcohol-induced glutamate hyperexcitability via mGluR5 blockade (Harris et al., 2002; Littleton and Zieglgansberger, 2003; Lominac et al., 2006). The present data provide the first evidence that glutamate activation of mGluR5 receptors located specifically within the NAC shell is necessary for high levels of binge alcohol drinking behavior under limited-access conditions and thereby point to NAC shell mGluR5 receptors as important for the “anti-alcohol” effects of systemic mGluR5 antagonist treatment – a finding consistent with the results of a recent intravenous alcohol self-administration study by Gass and Olive (2009). As the total protein expression of mGluR5 is unaltered within the NAC following either chronic, continuous alcohol drinking (Szumlinski et al., 2008b) or repeated bouts of binge alcohol drinking (Figure 3), it is possible that idiopathic or drug-induced anomalies in mGluR5 intracellular signaling might modulate the propensity to consume excessive amounts of alcohol in a manner independent of changes in receptor protein expression.
Consistent with the notion that mGluR5-mediated signaling contributes to an excessive alcohol drinking phenotype, the SHAC selected line exhibited a marked rise in the NAC Homer2a/b expression, relative to the SLAC selected line. Thus, an increase in this Homer isoform demonstrated previously to play an active role in regulating various aspects of alcohol reward in mice (Szumlinski et al., 2005b, 2008b) is a correlated response to selection. As observed upon mGluR5 blockade or mGluR5 deletion (see above), Homer2 deletion produces an alcohol-avoiding behavioral phenotype (Szumlinski et al., 2005b), which can be “rescued” by AAV-mediated restoration of NAC Homer2 expression. Moreover, AAV-mediated Homer2b over-expression within the NAC shell of both inbred B6 mice and B6-hybrid mice augments alcohol's rewarding/reinforcing properties (Szumlinski et al., 2005b, 2008b). However, mimicking the SHAC procedure-induced rise in NAC Homer2b levels by AAV-mediated Homer2 over-expression failed to augment alcohol intake in this binge alcohol-drinking model. The precise explanation for this negative result might relate to: (1) a ceiling effect upon alcohol drinking imposed by the limited access conditions of the SHAC procedure (although the alcohol intake of both AAV-treated groups in the NAC shell study was relatively low, compared to that of the NAC core and shRNA study; see Figure 4b); (2) competition for proline-rich motif binding between exogenous protein and the very high background of endogenously expressed Homer2 produced by repeated, binge alcohol intake (Figure 3); or (3) differences in the underlying motivational factors governing alcohol intake under limited access, forced-choice conditions. Nevertheless, the observation that shRNA-mediated knock-down of NAC Homer2 expression reduced binge alcohol-drinking (Figure 4b) in a manner consistent with the “anti-alcohol” phenotype of constitutive Homer2 knock-out mice (Szumlinski et al., 2005b), and akin to that observed in (1) mice treated intra-NAC with an mGluR5 antagonist (Figure 5) and (2) mice with a transgenic disruption of the Homer binding site on mGluR5 (Figure 6), supports an important role for mGluR5 signaling through Homer2 in excessive alcohol drinking. This notion is strengthened by the observation that an intra-NAC infusion of MPEP failed to reduce further the relatively low alcohol intake of mGluR5F1128R mutants (Figure 6). While not negating a potential role for Homer1 gene products in regulating binge alcohol drinking, this collection of data supports an important role for the physical interaction between mGluR5 and Homer2 in the “anti-binge” effects of mGluR5 antagonists.
Complementing earlier in vitro data (Rong et al., 2003) and supporting a potential role for PI3K activity in excessive alcohol intake, the SHAC procedure-induced rise in NAC Homer2a/b expression was accompanied by elevations in p(Tyr)p85α PI3K binding motifs (Figure 3), an index of PI3K activation (Zhang et al., 2006). Moreover, the basal NAC expression of p(Tyr)p85α PI3K binding motifs was elevated in a selectively bred mouse line exhibiting high “binge” alcohol intake (i.e., SHAC line; Figure 8) and reduced in mGluR5F1128R mutant mice exhibiting blunted binge drinking behavior (Figure 7). Pharmacological antagonism of PI3K activity within the NAC shell significantly reduced binge alcohol-drinking (estimated BACs ~ 0.83 mg/ml) and this effect was not additive with the reduction in drinking produced by mGluR5 inhibition (Figure 5) nor by transgenic inference of mGluR5-Homer binding (Figure 6). While not negating a potential role for Homer2 effects upon the conductance of voltage- and ligand-gated ion channels (e.g., the NMDA receptor), or Group1 mGluR-mediated activation of IP3-dependent calcium release or PKC activation (e.g., Tu et al., 1998; Kammermeier et al., 2000; Hwang et al., 2005; Mao et al., 2005; Sala et al., 2005; Szumlinski et al., 2005b; Yammamoto et al., 2005; Kammermeier and Worley, 2007; Kammermeier, 2008), the collection of data presented in this report, argues strongly in favor of increased mGluR5-mediated activation of PI3K, presumably via Homer2, as a critical molecular adaptation contributing to the propensity to binge drink excessive amounts of alcohol. Given the recent association of SNPs in PI3K with risky alcohol drinking behavior in adolescent humans (Desrivieres et al., 2008; Schumann et al., 2008), the present data have direct relevance not only for our basic understanding of how repeated bouts of excessive, binge alcohol drinking impact excitatory neurotransmission, but also inform as to potential gene candidates mediating vulnerability to this prevalent form of alcoholism.
This work was funded by NIH grants AA016650 (INIA West) and AA015351 to KKS, AA013478 (INIA West) to DAF, AA13519 (INIA West) and AA10760 to JCC, DA 00266 and DA011742 to PFW, as well as by grants from the Department of Veterans Affairs to DAF and JCC, and an Emerging Research Excellence Award, University of Auckland, to MK.