Previous studies showed that mice with genetic predisposition for high alcohol consumption as well as human alcoholics show changes in brain expression of genes related to immune signaling. In addition, mutant mice lacking genes related to immune function show decreased alcohol consumption (Blednov et al., in press), suggesting that immune signaling promotes alcohol consumption. To test the possibility that activation of immune signaling will increase alcohol consumption, we treated mice with lipopolysaccaride (LPS; 1 mg/kg, i.p.) and tested alcohol consumption in the continuous two-bottle choice test. To take advantage of the long-lasting activation of brain immune signaling by LPS, we measured drinking beginning one week or one month after LPS treatment and continued the studies for several months. LPS produced persistent increases in alcohol consumption in C57/Bl6 J (B6) inbred mice, FVBxB6F1 and B6xNZBF1 hybrid mice, but not in FVB inbred mice. To determine if this effect of LPS is mediated through binding to TLR4, we tested mice lacking CD14, a key component of TLR4 signaling. These null mutants showed no increase of alcohol intake after treatment with LPS. LPS treatment decreased ethanol-conditioned taste aversion but did not alter ethanol-conditioned place preference (B6xNZBF1 mice). Electro-physiological studies of dopamine neurons in the ventral tegmental area showed that pretreatment of mice with LPS decreased the neuronal firing rate. These results suggest that activation of immune signaling promotes alcohol consumption and alters certain aspects of alcohol reward/aversion.

Background Peroxisome proliferator-activated receptor (PPAR) agonists reduce voluntary ethanol consumption in rat models and are promising therapeutics in the treatment of drug addictions. We studied the effects of different classes of PPAR agonists on chronic ethanol intake and preference in mice with a genetic predisposition for high alcohol consumption and then examined human genome wide association data for polymorphisms in PPAR genes in alcohol-dependent subjects. Methods Two different behavioral tests were used to measure intake of 15% ethanol in C57BL/6J male mice: 24-hour two-bottle choice and limited access (3-hour) two-bottle choice, drinking in the dark. We measured the effects of pioglitazone (10 and 30 mg/kg), fenofibrate (50 and 150 mg/kg), GW0742 (10 mg/kg), tesaglitazar (1.5 mg/kg) and bezafibrate (25 and 75 mg/kg) on ethanol intake and preference. Fenofibric acid, the active metabolite of fenofibrate, was quantified in mouse plasma, liver, and brain by LC-MS/MS. Data from a human genome wide association study (GWAS) completed in the Collaborative Study on the Genetics of Alcoholism (COGA) was then used to analyze the association of single nucleotide polymorphisms (SNPs) in different PPAR genes (PPARA, PPARD, PPARG, and PPARGC1A) with two phenotypes: DSM-IV alcohol dependence (AD) and the DSM-IV criterion of withdrawal. Results Activation of two isoforms of PPARs, α and γ, reduced ethanol intake and preference in the two different consumption tests in mice. However, a selective PPARδ agonist or a pan agonist for all three PPAR isoforms did not decrease ethanol consumption. Fenofibric acid, the active metabolite of the PPARα agonist fenofibrate, was detected in liver, plasma, and brain after 1 or 8 days of oral treatment. The GWAS from COGA supported an association of SNPs in PPARA and PPARG with alcohol withdrawal and PPARGC1A with AD but found no association for PPARD with either phenotype. Conclusions We provide convergent evidence using both mouse and human data for specific PPARs in alcohol action. Reduced ethanol intake in mice and the genetic association between AD or withdrawal in humans highlight the potential for repurposing FDA-approved PPARα or PPARγ agonists for the treatment of AD.

Some anti-inflammatory medications reduce alcohol consumption in rodent models. Inhibition of phosphodiesterases (PDE) increases cAMP and reduces inflammatory signaling. Rolipram, an inhibitor of PDE4, markedly reduced ethanol intake and preference in mice and reduced ethanol seeking and consumption in alcohol-preferring fawn-hooded rats (Hu et al., 2011; Wen et al., 2012). To determine if these effects were specific for PDE4, we compared nine PDE inhibitors with different subtype selectivity: propentofylline (nonspecific), vinpocetine (PDE1), olprinone, milrinone (PDE3), zaprinast (PDE5), rolipram, mesopram, piclamilast, and CDP840 (PDE4). Alcohol intake was measured in C57BL/6J male mice using 24-h two-bottle choice and two-bottle choice with limited (3-h) access to alcohol. Only the selective PDE4 inhibitors reduced ethanol intake and preference in the 24-h two-bottle choice test. For rolipram, piclamilast, and CDP840, this effect was observed after the first 6 h but not after the next 18 h. Mesopram, however, produced a long-lasting reduction of ethanol intake and preference. In the limited access test, rolipram, piclamilast, and mesopram reduced ethanol consumption and total fluid intake and did not change preference for ethanol, whereas CDP840 reduced both consumption and preference without altering total fluid intake. Our results provide novel evidence for a selective role of PDE4 in regulating ethanol drinking in mice. We suggest that inhibition of PDE4 may be an unexplored target for medication development to reduce excessive alcohol consumption.

184 -191, 2008). The goal of our study was to determine the role of receptors containing this subunit in alcohol action. We designed an ␣2 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity in a recombinant expression system. Knockin mice containing this mutant subunit were tested in a range of ethanol behavioral tests. These mutant mice did not develop the typical conditioned taste aversion in response to ethanol and showed complete loss of the motor stimulant effects of ethanol. Conversely, they also demonstrated changes in ethanol intake and preference in multiple tests. The knockin mice showed increased ethanolinduced hypnosis but no difference in anxiolytic effects or recovery from acute ethanol-induced motor incoordination.Overall, these studies demonstrate that the effects of ethanol at GABAergic synapses containing the ␣2 subunit are important for specific behavioral effects of ethanol that may be relevant to the genetic linkage of this subunit with human alcoholism.

GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.

Background In our companion article, we examined the role of MyD88-dependent signaling in ethanol consumption in mice lacking key components of this inflammatory pathway and observed differential effects on drinking. Here we studied the role of these same signaling components in the acute sedative, intoxicating, and physiological effects of ethanol. TLR4 has been reported to strongly reduce the duration of ethanol-induced sedation, though most studies do not support its direct involvement in ethanol consumption. We examined TLR4 and other MyD88 pathway molecules to determine signaling specificity in acute ethanol-related behaviors. We also studied other GABAergic sedatives to gauge the ethanol specificity and potential role for GABA in ethanol’s sedative and intoxicating effects in the mutant mice. Methods Loss of righting reflex (LORR) and recovery from motor incoordination were studied following acute injection of ethanol or other sedative drugs in male and female control C57BL/6J mice vs. mice lacking CD14, TLR2, TLR4 (C57BL/10ScN), or MyD88. We also examined ethanol-induced hypothermia and blood ethanol clearance in these mice. Results Male and female mice lacking TLR4 or MyD88 showed reduced duration of ethanol-induced LORR and faster recovery from ethanol-induced motor incoordination. MyD88 KO mice had slightly faster recovery from ethanol-induced hypothermia compared to control mice. None of the mutants differed from control mice in the rate of blood ethanol clearance. There were no genotype differences in the duration of gaboxadol-induced LORR, and only mice lacking TLR4 were less sensitive to the sedative effects of pentobarbital. Faster recovery from diazepam-induced motor incoordination was observed in CD14, TLR4, and MyD88 null mice of both sexes. Conclusions TLR4 and MyD88 were key mediators of the sedative and intoxicating effects of ethanol and GABAergic sedatives, indicating a strong influence of TLR4-MyD88 signaling on GABAergic function. Despite the involvement of TLR4 in ethanol’s acute behaviors, it did not regulate ethanol consumption in any drinking model as shown in our companion article. Collectively, our studies demonstrate differential effects of TLR-MyD88 components in the acute vs. chronic actions of ethanol.

Glycine receptors (GlyRs) are broadly expressed in the central nervous system. Ethanol enhances the function of brain GlyRs, and the GlyRa1 subunit is associated with some of the behavioral actions of ethanol, such as loss of righting reflex. The in vivo role of GlyRa2 and a3 subunits in alcohol responses has not been characterized despite high expression levels in the nucleus accumbens and amygdala, areas that are important for the rewarding properties of drugs of abuse. We used an extensive panel of behavioral tests to examine ethanol actions in mice lacking Glra2 (the gene encoding the glycine receptor alpha 2 subunit) or Glra3 (the gene encoding the glycine receptor alpha 3 subunit). Deletion of Glra2 or Glra3 alters specific ethanol-induced behaviors. Glra2 knockout mice demonstrate reduced ethanol intake and preference in the 24-hour two-bottle choice test and increased initial aversive responses to ethanol and lithium chloride. In contrast, Glra3 knockout mice show increased ethanol intake and preference in the 24-hour intermittent access test and increased development of conditioned taste aversion to ethanol. Mutants and wild-type mice consumed similar amounts of ethanol in the limited access drinking in the dark test. Other ethanol effects, such as anxiolysis, motor incoordination, loss of righting reflex, and acoustic startle response, were not altered in the mutants. The behavioral changes in mice lacking GlyRa2 or a3 subunits were distinct from effects previously observed in mice with knock-in mutations in the a1 subunit. We provide evidence that GlyRa2 and a3 subunits may regulate ethanol consumption and the aversive response to ethanol.

Background Several peroxisome proliferator-activated receptor (PPAR) agonists reduce voluntary alcohol consumption in rodent models, and evidence suggests that PPARα and γ subunits play an important role in this effect. To define the subunit dependence of this action, we tested selective PPARα and α/γ agonists and antagonists in addition to null mutant mice lacking PPARα. Methods The effects of fenofibrate (PPARα agonist) and tesaglitazar (PPARα/γ agonist) on continuous and intermittent two-bottle choice drinking tests were examined in male and female wild-type mice and in male mice lacking PPARα. We compared the ability of MK886 (PPARα antagonist) and GW9662 (PPARγ antagonist) to inhibit the effects of fenofibrate and tesaglitazar in wild-type mice. The estrogen receptor antagonist, tamoxifen, can inhibit PPARγ-dependent transcription and was also studied in male and female mice. Results Fenofibrate and tesaglitazar reduced ethanol consumption and preference in wild-type mice, but these effects were not observed in mice lacking PPARα. MK886 inhibited the action of fenofibrate, but not tesaglitazer, while GW9662 did not inhibit either agonist. The PPAR agonists were more effective in male mice compared to females, and drinking in the continuous two-bottle choice test was more sensitive to fenofibrate and tesaglitazar compared to drinking in the intermittent access test. Tamoxifen also reduced ethanol consumption in male mice and this action was inhibited by GW9662, but not MK886, suggesting that it acts by activation of PPARγ. Conclusions Our study using selective PPAR agonists, antagonists, and null mutant mice indicates a key role for PPARα in mediating reduced ethanol consumption by fenofibrate and tesaglitazar.