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.

Deletion of Tlr3 reduces acute tolerance to alcohol and alcohol consumption in the intermittent access procedure in male mice.

Pharmacological studies implicate toll-like receptor 3 (TLR3) signaling in alcohol drinking. We examined the role of TLR3 in behavioral responses to alcohol and GABAergic drugs by studying Tlr3 -/- mice. Because of opposing signaling between TLR3 and MyD88 pathways, we also evaluated Myd88 -/- mice. Ethanol consumption and preference decreased in male but not in female Tlr3 -/- mice during two-bottle choice every-other-day (2BC-EOD) drinking. There were no genotype differences in either sex during continuous or limited-access drinking. Null mutations in Tlr3 or Myd88 did not alter conditioned taste aversion to alcohol and had small or no effects on conditioned place preference. The Tlr3 null mutation did not alter acute alcohol withdrawal. Male, but not female, Tlr3 -/- mice took longer than wild-type littermates to recover from ataxia by ethanol or diazepam and longer to recover from sedative-hypnotic effects of ethanol or gaboxadol, indicating regulation of GABAergic signaling by TLR3. Acute functional tolerance (AFT) to alcohol-induced ataxia was decreased in Tlr3 -/- mice but was increased in Myd88 -/- mice. Thus, MyD88 and TLR3 pathways coordinately regulate alcohol consumption and tolerance to intoxicating doses of alcohol and GABAergic drugs. Despite similar alcohol metabolism and similar amounts of total alcohol consumed during 2BC and 2BC-EOD procedures in C57BL/6J mice, only 2BC-EOD drinking induced tolerance to alcohol-induced ataxia. Ataxia recovery was inversely correlated with level of drinking in wild-type and Tlr3 -/- littermates. Thus, deleting Tlr3 reduces alcohol consumption by reducing AFT to alcohol and not by altering tolerance induced by 2BC-EOD drinking.

Apremilast regulates acute effects of ethanol and other GABAergic drugs via protein kinase A-dependent signaling.

Phosphodiesterase type 4 (PDE4) inhibitors prevent hydrolysis of cyclic adenosine monophosphate and increase protein kinase A (PKA)-mediated phosphorylation. PDE4 inhibitors also regulate responses to ethanol and GABAergic drugs. We investigated mechanisms by which the PDE4 inhibitor, apremilast, regulates acute effects of ethanol and GABAergic drugs in male and female mice. Apremilast prolonged the sedative-hypnotic effects of gaboxadol, zolpidem, and propofol but did not alter etomidate effects, and unexpectedly shortened the sedative-hypnotic effects of diazepam. Apremilast prolonged rotarod ataxia induced by zolpidem, propofol, and loreclezole, shortened recovery from diazepam, but had no effect on ataxia induced by gaboxadol or etomidate. The PKA inhibitor H-89 blocked apremilast's ability to prolong the sedative-hypnotic effects of ethanol, gaboxadol, and propofol and to prolong ethanol- and propofol-induced ataxia. H-89 also blocked apremilast's ability to shorten the sedative-hypnotic and ataxic effects of diazepam. The ß1-specific antagonist, salicylidene salicylhydrazide (SCS), produced faster recovery from ethanol- and diazepam-induced ataxia, but did not alter propofol- or etomidate-induced ataxia. SCS shortened the sedative-hypnotic effects of ethanol and diazepam but not of propofol. In Xenopus oocytes, a phosphomimetic (aspartate) mutation at the PKA phosphorylation site in ß1 subunits decreased the maximal GABA current in receptors containing α1 or α3, but not α2 subunits. In contrast, phosphomimetic mutations at PKA sites in ß3 subunits increased the maximal GABA current in receptors containing α1 or α2, but not α3 subunits. The GABA potency and allosteric modulation by ethanol, propofol, etomidate, zolpidem, flunitrazepam, or diazepam were not altered by these mutations. We propose a model whereby apremilast increases PKA-mediated phosphorylation of ß1-and ß3-containing GABAA receptors and selectively alters acute tolerance to ethanol and GABAergic drugs.