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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., (Copyright © 2017 by the Research Society on Alcoholism.)

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., (Copyright © 2014 by the Research Society on Alcoholism.)