The PDE4 inhibitor apremilast modulates ethanol responses in Gabrb1-S409A knock-in mice via PKA-dependent and independent mechanisms.

We previously showed that the PDE4 inhibitor apremilast reduces ethanol consumption in mice by protein kinase A (PKA) and GABAergic mechanisms. Preventing PKA phosphorylation of GABAA ß3 subunits partially blocked apremilast-mediated decreases in drinking. Here, we produced Gabrb1-S409A mice to render GABAA ß1 subunits resistant to PKA-mediated phosphorylation. Mass spectrometry confirmed the presence of the S409A mutation and lack of changes in ß1 subunit expression or phosphorylation at other residues. ß1-S409A male and female mice did not differ from wild-type C57BL/6J mice in expression of Gabrb1, Gabrb2, or Gabrb3 subunits or in behavioral characteristics. Apremilast prolonged recovery from ethanol ataxia to a greater extent in Gabrb1-S409A mice but prolonged recovery from zolpidem and propofol to a similar extent in both genotypes. Apremilast shortened recovery from diazepam ataxia in wild-type but prolonged recovery in Gabrb1-S409A mice. In wild-type mice, the PKA inhibitor H89 prevented apremilast modulation of ataxia by ethanol and diazepam, but not by zolpidem. In Gabrb1-S409A mice, inhibiting PKA or EPAC2 (exchange protein directly activated by cAMP) partially reversed apremilast potentiation of ethanol, diazepam, and zolpidem ataxia. Apremilast prevented acute tolerance to ethanol ataxia in both genotypes, but there were no genotype differences in ethanol consumption before or after apremilast. In contrast to results in Gabrb3-S408A/S409A mice, PKA phosphorylation of ß1-containing GABAA receptors is not required for apremilast's effects on acute tolerance or on ethanol consumption but is required for its ability to decrease diazepam intoxication. Besides PKA we identified EPAC2 as an additional cAMP-dependent mechanism by which apremilast regulates responses to GABAergic drugs.

Knockdown of Tlr3 in dorsal striatum reduces ethanol consumption and acute functional tolerance in male mice.

Systemic activation of toll-like receptor 3 (TLR3) signaling using poly(I:C), a TLR3 agonist, drives ethanol consumption in several rodent models, while global knockout of Tlr3 reduces drinking in C57BL/6J male mice. To determine if brain TLR3 pathways are involved in drinking behavior, we used CRISPR/Cas9 genome editing to generate a Tlr3 floxed (Tlr3F/F) mouse line. After sequence confirmation and functional validation of Tlr3 brain transcripts, we injected Tlr3F/F male mice with an adeno-associated virus expressing Cre recombinase (AAV5-CMV-Cre-GFP) to knockdown Tlr3 in the medial prefrontal cortex, nucleus accumbens, or dorsal striatum (DS). Only Tlr3 knockdown in the DS decreased two-bottle choice, every-other-day (2BC-EOD) ethanol consumption. DS-specific deletion of Tlr3 also increased intoxication and prevented acute functional tolerance to ethanol. In contrast, poly(I:C)-induced activation of TLR3 signaling decreased intoxication in male C57BL/6J mice, consistent with its ability to increase 2BC-EOD ethanol consumption in these mice. We also found that TLR3 was highly colocalized with DS neurons. AAV5-Cre transfection occurred predominantly in neurons, but there was minimal transfection in astrocytes and microglia. Collectively, our previous and current studies show that activating or inhibiting TLR3 signaling produces opposite effects on acute responses to ethanol and on ethanol consumption. While previous studies, however, used global knockout or systemic TLR3 activation (which alter peripheral and brain innate immune responses), the current results provide new evidence that brain TLR3 signaling regulates ethanol drinking. We propose that activation of TLR3 signaling in DS neurons increases ethanol consumption and that a striatal TLR3 pathway is a potential target to reduce excessive drinking.

Long-term ethanol exposure: Temporal pattern of microRNA expression and associated mRNA gene networks in mouse brain.

Long-term alcohol use can result in lasting changes in brain function, ultimately leading to alcohol dependence. These functional alterations arise from dysregulation of complex gene networks, and growing evidence implicates microRNAs as key regulators of these networks. We examined time- and brain region-dependent changes in microRNA expression after chronic intermittent ethanol (CIE) exposure in C57BL/6J mice. Animals were sacrificed at 0, 8, and 120h following the last exposure to four weekly cycles of CIE vapor and we measured microRNA expression in prefrontal cortex (PFC), nucleus accumbens (NAC), and amygdala (AMY). The number of detected (395-419) and differentially expressed (DE, 42-47) microRNAs was similar within each brain region. However, the DE microRNAs were distinct among brain regions and across time within each brain region. DE microRNAs were linked with their DE mRNA targets across each brain region. In all brain regions, the greatest number of DE mRNA targets occurred at the 0 or 8h time points and these changes were associated with microRNAs DE at 0 or 8h. Two separate approaches (discrete temporal association and hierarchical clustering) were combined with pathway analysis to further characterize the temporal relationships between DE microRNAs and their 120h DE targets. We focused on targets dysregulated at 120h as this time point represents a state of protracted withdrawal known to promote an increase in subsequent ethanol consumption. Discrete temporal association analysis identified networks with highly connected genes including ERK1/2 (mouse equivalent Mapk3, Mapk1), Bcl2 (in AMY networks) and Srf (in PFC networks). Similarly, the cluster-based analysis identified hub genes that include Bcl2 (in AMY networks) and Srf in PFC networks, demonstrating robust microRNA-mRNA network alterations in response to CIE exposure. In contrast, datasets utilizing targets from 0 and 8h microRNAs identified NF-kB-centered networks (in NAC and PFC), and Smad3-centered networks (in AMY). These results demonstrate that CIE exposure results in dynamic and complex temporal changes in microRNA-mRNA gene network structure.

Mutation of the inhibitory ethanol site in GABAA ρ1 receptors promotes tolerance to ethanol-induced motor incoordination.

Genes encoding the ρ1/2 subunits of GABAA receptors have been associated with alcohol (ethanol) dependence in humans, and ρ1 was also shown to regulate some of the behavioral effects of ethanol in animal models. Ethanol inhibits GABA-mediated responses in wild-type (WT) ρ1, but not ρ1(T6'Y) mutant receptors expressed in Xenopus laevis oocytes, indicating the presence of an inhibitory site for ethanol in the second transmembrane helix. In this study, we found that ρ1(T6'Y) receptors expressed in oocytes display overall normal responses to GABA, the endogenous GABA modulator (zinc), and partial agonists (ß-alanine and taurine). We generated ρ1 (T6'Y) knockin (KI) mice using CRISPR/Cas9 to test the behavioral importance of the inhibitory actions of ethanol on this receptor. Both ρ1 KI and knockout (KO) mice showed faster recovery from acute ethanol-induced motor incoordination compared to WT mice. Both KI and KO mutant strains also showed increased tolerance to motor impairment produced by ethanol. The KI mice did not differ from WT mice in other behavioral actions, including ethanol intake and preference, conditioned taste aversion to ethanol, and duration of ethanol-induced loss of righting reflex. WT and KI mice did not differ in levels of ρ1 or ρ2 mRNA in cerebellum or in ethanol clearance. Our findings indicate that the inhibitory site for ethanol in GABAA ρ1 receptors regulates acute functional tolerance to moderate ethanol intoxication. We note that low sensitivity to alcohol intoxication has been linked to risk for development of alcohol dependence in humans.

Chronic ethanol exposure produces time- and brain region-dependent changes in gene coexpression networks.

Repeated ethanol exposure and withdrawal in mice increases voluntary drinking and represents an animal model of physical dependence. We examined time- and brain region-dependent changes in gene coexpression networks in amygdala (AMY), nucleus accumbens (NAC), prefrontal cortex (PFC), and liver after four weekly cycles of chronic intermittent ethanol (CIE) vapor exposure in C57BL/6J mice. Microarrays were used to compare gene expression profiles at 0-, 8-, and 120-hours following the last ethanol exposure. Each brain region exhibited a large number of differentially expressed genes (2,000-3,000) at the 0- and 8-hour time points, but fewer changes were detected at the 120-hour time point (400-600). Within each region, there was little gene overlap across time (~20%). All brain regions were significantly enriched with differentially expressed immune-related genes at the 8-hour time point. Weighted gene correlation network analysis identified modules that were highly enriched with differentially expressed genes at the 0- and 8-hour time points with virtually no enrichment at 120 hours. Modules enriched for both ethanol-responsive and cell-specific genes were identified in each brain region. These results indicate that chronic alcohol exposure causes global 'rewiring' of coexpression systems involving glial and immune signaling as well as neuronal genes.

Glycine receptors containing α2 or α3 subunits regulate specific ethanol-mediated behaviors.

Glycine receptors (GlyRs) are broadly expressed in the central nervous system. Ethanol enhances the function of brain GlyRs, and the GlyRα1 subunit is associated with some of the behavioral actions of ethanol, such as loss of righting reflex. The in vivo role of GlyRα2 and α3 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 GlyRα2 or α3 subunits were distinct from effects previously observed in mice with knock-in mutations in the α1 subunit. We provide evidence that GlyRα2 and α3 subunits may regulate ethanol consumption and the aversive response to ethanol.

GABAA receptors containing ρ1 subunits contribute to in vivo effects of ethanol in mice.

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.

Gene expression in brain and liver produced by three different regimens of alcohol consumption in mice: comparison with immune activation.

Chronically available alcohol escalates drinking in mice and a single injection of the immune activator lipopolysaccharide can mimic this effect and result in a persistent increase in alcohol consumption. We hypothesized that chronic alcohol drinking and lipopolysaccharide injections will produce some similar molecular changes that play a role in regulation of alcohol intake. We investigated the molecular mechanisms of chronic alcohol consumption or lipopolysaccharide insult by gene expression profiling in prefrontal cortex and liver of C57BL/6J mice. We identified similar patterns of transcriptional changes among four groups of animals, three consuming alcohol (vs water) in different consumption tests and one injected with lipopolysaccharide (vs. vehicle). The three tests of alcohol consumption are the continuous chronic two bottle choice (Chronic), two bottle choice available every other day (Chronic Intermittent) and limited access to one bottle of ethanol (Drinking in the Dark). Gene expression changes were more numerous and marked in liver than in prefrontal cortex for the alcohol treatments and similar in the two tissues for lipopolysaccharide. Many of the changes were unique to each treatment, but there was significant overlap in prefrontal cortex for Chronic-Chronic Intermittent and for Chronic Intermittent-lipopolysaccharide and in liver all pairs showed overlap. In silico cell-type analysis indicated that lipopolysaccharide had strongest effects on brain microglia and liver Kupffer cells. Pathway analysis detected a prefrontal cortex-based dopamine-related (PPP1R1B, DRD1, DRD2, FOSB, PDNY) network that was highly over-represented in the Chronic Intermittent group, with several genes from the network being also regulated in the Chronic and lipopolysaccharide (but not Drinking in the Dark) groups. Liver showed a CYP and GST centered metabolic network shared in part by all four treatments. We demonstrate common consequences of chronic alcohol consumption and immune activation in both liver and brain and show distinct genomic consequences of different types of alcohol consumption.