The mammalian circadian clock exhibits chronic tolerance to alcohol in vitro

Alcohol abuse leads to cognitive defects, depression, and sleep disturbances. The connection between sleep and circadian disruptions led us to investigate ethanol's effect on the circadian clock. We demonstrated that acute ethanol blocks photic phase shifts in vivo and glutamatergic phase shifts in vitro. Ethanol exposure can lead to acute, rapid and/or chronic tolerance. We determined that the suprachiasmatic nucleus (SCN) circadian clock exhibits acute (developing in <;30 min) tolerance in vitro and rapid (develops in 8-24 h) tolerance in vitro and in vivo. We are currently investigating chronic tolerance. C57BL/6 mice are given access to 15% ethanol between zeitgeber time (ZT) 11 (where ZT 0 = lights-on and ZT 12 = lights off) and ZT 15 for 10 days. SCN brain slices were made the following morning and treated at either ZT 16 or ZT 23 with glutamate (1mM) + ethanol for 10 min. The next day SCN neuronal activity was monitored to determine the time of peak activity. 20mM ethanol blocks glutamatergic phase shifts in tissue from ethanol-naïve mice, while 200mM ethanol is needed in tissue from ethanol-exposed mice. These data suggest a similar shift in effective concentration as that seen during rapid tolerance. Currently we are investigating potential changes in surface expression of NR2B across the circadian cycle (at ZT 6, ZT 16, and ZT 23) and in response to ethanol consumption. Preliminary data suggests that surface expression of NR2B is highest at ZT 16 and lowest at ZT 23. Future experiments will expand on these studies.