A neuropeptide signal confers ethanol state dependency during olfactory learning in Caenorhabditis elegans.

Alcohol intoxication can impact learning and this may contribute to the development of problematic alcohol use. In alcohol (ethanol)-induced state-dependent learning (SDL), information learned while an animal is intoxicated is recalled more effectively when the subject is tested while similarly intoxicated than if tested while not intoxicated. When Caenorhabditis elegans undergoes olfactory learning (OL) while intoxicated, the learning becomes state dependent such that recall of OL is only apparent if the animals are tested while intoxicated. We found that two genes known to be required for signal integration, the secreted peptide HEN-1 and its receptor tyrosine kinase, SCD-2, are required for SDL. Expression of hen-1 in the ASER neuron and scd-2 in the AIA neurons was sufficient for their functions in SDL. Optogenetic activation of ASER in the absence of ethanol during learning could confer ethanol state dependency, indicating that ASER activation is sufficient to signal ethanol intoxication to the OL circuit. To our surprise, ASER activation during testing did not substitute for ethanol intoxication, demonstrating that the effects of ethanol on learning and recall rely on distinct signals. Additionally, intoxication-state information could be added to already established OL, but state-dependent OL did not lose state information when the intoxication signal was removed. Finally, dopamine is required for state-dependent OL, and we found that the activation of ASER cannot bypass this requirement. Our findings provide a window into the modulation of learning by ethanol and suggest that ethanol acts to modify learning using mechanisms distinct from those used during memory access.

SWI/SNF complexes act through CBP-1 histone acetyltransferase to regulate acute functional tolerance to alcohol.

BACKGROUND: SWI/SNF chromatin remodeling genes are required for normal acute responses to alcohol in C. elegans and are associated with alcohol use disorder in two human populations. In an effort to discover the downstream genes that are mediating this effect, we identified SWI/SNF-regulated genes in C. elegans. RESULTS: To identify SWI/SNF-regulated genes in adults, we compared mRNA expression in wild type and swsn-1(os22ts) worms under conditions that produce inactive swsn-1 in mature cells. To identify SWI/SNF-regulated genes in neurons, we compared gene expression in swsn-9(ok1354) null mutant worms that harbor a neuronal rescue or a control construct. RNA sequencing was performed to an average depth of 25 million reads per sample using 50-base, paired-end reads. We found that 6813 transcripts were significantly differentially expressed between swsn-1(os22ts) mutants and wild-type worms and 2412 transcripts were significantly differentially expressed between swsn-9(ok1354) mutants and swsn-9(ok1354) mutants with neuronal rescue. We examined the intersection between these two datasets and identified 603 genes that were differentially expressed in the same direction in both comparisons; we defined these as SWI/SNF-regulated genes in neurons and in adults. Among the differentially expressed genes was cbp-1, a C. elegans homolog of the mammalian CBP/p300 family of histone acetyltransferases. CBP has been implicated in the epigenetic regulation in response to alcohol in animal models and a polymorphism in the human CBP gene, CREBBP, has been associated with alcohol-related phenotypes. We found that cbp-1 is required for the development of acute functional tolerance to alcohol in C. elegans. CONCLUSIONS: We identified 603 transcripts that were regulated by two different SWI/SNF complex subunits in adults and in neurons. The SWI/SNF-regulated genes were highly enriched for genes involved in membrane rafts, suggesting an important role for this membrane microdomain in the acute alcohol response. Among the differentially expressed genes was cbp-1; CBP-1 homologs have been implicated in alcohol responses across phyla and we found that C. elegans cbp-1 was required for the acute alcohol response in worms.

The Mammalian Circadian Clock Exhibits Chronic Ethanol Tolerance and Withdrawal-Induced Glutamate Hypersensitivity, Accompanied by Changes in Glutamate and TrkB Receptor Proteins.

BACKGROUND: Alcohol tolerance and withdrawal-induced effects are criteria for alcohol use disorders listed by the DSM-V. Although tolerance and withdrawal have been studied over many decades, there is still uncertainty regarding mechanistic distinctions that characterize these different forms of ethanol (EtOH)-induced plasticity. Previously, we demonstrated that the suprachiasmatic nucleus (SCN) circadian clock develops both acute and rapid tolerance to EtOH inhibition of glutamate-induced circadian phase shifts. Here, we demonstrate that chronic EtOH tolerance and withdrawal-induced glutamate hypersensitivity occur in vitro and that rapid tolerance, chronic tolerance, and glutamate hypersensitivity have distinct cellular changes. METHODS: We use single-unit extracellular electrophysiological recordings to determine whether chronic tolerance to EtOH inhibition of glutamatergic phase shifts and withdrawal-induced glutamate hypersensitivity develop in the SCN. We use Western blotting to compare phosphorylation state and total expression of N-methyl-D-aspartate (NMDA) receptor subunits and associated proteins in the SCN after mice were exposed to varying EtOH consumption paradigms. RESULTS: Chronic tolerance developed after a minimum of 8 days of 4 h/d EtOH access, as indicated by a decreased sensitivity to EtOH inhibition of glutamate-induced phase shifts. We also observed an increased sensitivity to glutamate-induced phase shifts in SCN tissue following withdrawal. We demonstrated an increase in the ratio of NR2B:NR2A NMDA receptor subunit expression after 21 days, but not after 10 days of EtOH drinking. This increase persisted during EtOH withdrawal, along with an increase in NR2B Y1472 phosphorylation, mature brain-derived neurotrophic factor, and phosphorylated TrkB. CONCLUSIONS: These results demonstrate that multiple tolerance forms and withdrawal-induced glutamate hypersensitivity occur in the SCN and that these different forms of EtOH-induced plasticity are accompanied by distinct changes in cellular physiology. Importantly, this study further demonstrates the power of using the SCN as a model system to investigate EtOH-induced plasticity.

Meta-Analysis of Genetic Influences on Initial Alcohol Sensitivity.

BACKGROUND: Previous studies indicate that low initial sensitivity to alcohol may be a risk factor for later alcohol misuse. Evidence suggests that initial sensitivity is influenced by genetic factors, but few molecular genetic studies have been reported. METHODS: We conducted a meta-analysis of 2 population-based genome-wide association studies of the Self-Rating of the Effects of Alcohol scale. Our final sample consisted of 7,339 individuals (82.3% of European descent; 59.2% female) who reported having used alcohol at least 5 times. In addition, we estimated single nucleotide polymorphism (SNP)-based heritability and conducted a series of secondary aggregate genetic analyses. RESULTS: No individual locus reached genome-wide significance. Gene and set based analyses, both overall and using tissue-specific expression data, yielded largely null results, and genes previously implicated in alcohol problems and consumption were overall not associated with initial sensitivity. Only 1 gene set, related to hormone signaling and including core clock genes, survived correction for multiple testing. A meta-analysis of SNP-based heritability resulted in a modest estimate of hSNP2  = 0.19 (SE = 0.10), though this was driven by 1 sample (N = 3,683, hSNP2  = 0.36, SE = 0.14, p = 0.04). No significant genetic correlations with other relevant outcomes were observed. CONCLUSIONS: Findings yielded only modest support for a genetic component underlying initial alcohol sensitivity. Results suggest that its biological underpinnings may diverge somewhat from that of other alcohol outcomes and may be related to core clock genes or other aspects of hormone signaling. Larger samples, ideally of prospectively assessed samples, are likely necessary to improve gene identification efforts and confirm the current findings.

The mammalian circadian clock in the suprachiasmatic nucleus exhibits rapid tolerance to ethanol in vivo and in vitro.

BACKGROUND: Ethanol (EtOH) triggers cellular adaptations that induce tolerance in many brain areas, including the suprachiasmatic nucleus (SCN), the site of the master circadian clock. EtOH inhibits light-induced phase shifts in the SCN in vivo and glutamate-induced phase shifts in vitro. The in vitro phase shifts develop acute tolerance to EtOH, occurring within minutes of initial exposure, while the in vivo phase shifts exhibit no evidence of chronic tolerance. An intermediate form, rapid tolerance, is not well studied but may predict subsequent chronic tolerance. Here, we investigated rapid tolerance in the SCN clock. METHODS: Adult C57BL/6 mice were provided 15% EtOH or water for one 12-hour lights-off period. For in vitro experiments, SCN-containing brain slices were prepared in the morning and treated for 10 minutes with glutamate +/- EtOH the following night. Single-cell neuronal firing rates were recorded extracellularly during the subsequent day to determine SCN clock phase. For in vivo experiments, mice receiving EtOH 24 hours previously were exposed to a 30-minute light pulse immediately preceded by intraperitoneal saline or 2 g/kg EtOH injection. Mice were then placed in constant darkness and their phase-shifting responses measured. RESULTS: In vitro, the SCN clock from EtOH-exposed mice exhibited rapid tolerance, with a 10-fold increase in EtOH needed to inhibit glutamate-induced phase shifts. Co-application of brain-derived neurotrophic factor prevented EtOH inhibition, consistent with experiments using EtOH-naïve mice. Rapid tolerance lasts 48 to 96 hours, depending on whether assessing in vitro phase advances or phase delays. Similarly, in vivo, prior EtOH consumption prevented EtOH's acute blockade of photic phase delays. Finally, immunoblot experiments showed no changes in SCN glutamate receptor subunit (NR2B) expression or phosphorylation in response to rapid tolerance induction. CONCLUSIONS: The SCN circadian clock develops rapid tolerance to EtOH as assessed both in vivo and in vitro, and the tolerance lasts for several days. These data demonstrate the utility of the circadian system as a model for investigating cellular mechanisms through which EtOH acts in the brain.