J. Neurochem. (2010) 115, 782–794. Abstract Insomnia is a severe symptom of alcohol withdrawal; however, the underlying neuronal mechanism is yet unknown. We hypothesized that chronic ethanol exposure will impair basal forebrain (BF) adenosinergic mechanism resulting in insomnia‐like symptoms. We performed a series of experiments in Sprague–Dawley rats to test our hypothesis. We used Majchrowicz’s chronic binge ethanol protocol to induce ethanol dependency. Our first experiment verified the effects of ethanol withdrawal on sleep–wakefulness. Significant increase in wakefulness was observed during ethanol withdrawal. Next, we examined c‐Fos expression (marker of neuronal activation) in BF wake‐promoting neurons during ethanol withdrawal. There was a significant increase in the number of BF wake‐promoting neurons with c‐Fos immunoreactivity. Our third experiment examined the effects of ethanol withdrawal on sleep deprivation induced increase in BF adenosine levels. Sleep deprivation did not increase BF adenosine levels in ethanol dependent rats. Our last experiment examined the effects of ethanol withdrawal on equilibrative nucleoside transporter 1 and A1 receptor expression in the BF. There was a significant reduction in A1 receptor and equilibrative nucleoside transporter 1 expression in the BF of ethanol dependent rats. Based on these results, we suggest that insomnia observed during ethanol withdrawal is caused because of impaired adenosinergic mechanism in the BF.

BACKGROUND Ethanol intake has significant impact on sleep. However, the cellular substrates responsible for sleep promotion following ethanol intake are unknown. The purine nucleoside, adenosine, is responsible for mediating many neuronal and behavioral responses to ethanol. Studies performed in cell cultures suggest that ethanol inhibits equilibrative nucleoside transporter 1 to block the reuptake of adenosine resulting in increased extracellular adenosine. Adenosine also has a pivotal role in sleep regulation. Adenosine acts via A1 receptor to inhibit the wake-promoting neurons of the basal forebrain (BF) resulting in the promotion of sleep. Is ethanol induced sleep associated with the inhibition of the BF wake-promoting neurons? Do adenosinergic mechanisms in the BF have a role in sleep-promoting effects of ethanol? METHODS To address these questions, we performed three experiments in Sprague Dawley rats. First, we verified the effect of ethanol on sleep promotion. Second, we evaluated the effect of ethanol on c-Fos expression (a marker of neuronal activation) in the BF wake-promoting neurons and third we monitored the effects of A1 receptor blockade in the BF on ethanol induced sleep. RESULTS Significant increase in non-rapid eye movement (NREM) sleep with a concomitant decrease in wakefulness was observed during first 12 hours post- ethanol. REM sleep remained unaffected. Ethanol administration caused a significant decrease in the number of BF wake-promoting neurons with c-Fos immunoreactivity. Bilateral microinjections of a selective A1R receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine into the BF significantly attenuated sleep-promoting effects of ethanol. CONCLUSION These results suggest that the inhibition of BF wake-promoting neurons by adenosinergic mechanism may be responsible for the sleep promoting effects of ethanol. We believe our study is the first to investigate the cellular mechanisms responsible for the somnogenic effects of ethanol.

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