A single episode of binge alcohol drinking causes sleep disturbance, disrupts sleep homeostasis, and down-regulates equilibrative nucleoside transporter 1.

Binge alcohol drinking, a risky pattern of alcohol consumption, has severe consequences toward health and well-being of an individual, his family, and society. Although, binge drinking has detrimental effects on sleep, underlying mechanisms are unknown. We used adult male C57BL/6J mice and exposed them to a single, 4-h session of binge alcohol self-administration, in stress-free environment, to examine neuronal mechanisms affecting sleep. We first verified binge pattern of alcohol consumption. When allowed to self-administer alcohol in a non-stressful environment, mice consumed alcohol in a binge pattern. Next, effect of binge drinking on sleep-wakefulness was monitored. While sleep-wakefulness remained unchanged during drinking session, significant increase in non-rapid eye movement (NREM) sleep was observed during 4 h of active period post-binge, followed by increased wakefulness, reduced sleep during subsequent sleep (light) period; although the timing of sleep onset (at lights-on) remained unaffected. Next, electrophysiological and biochemical indicators of sleep homeostasis were examined using sleep deprivation-recovery sleep paradigm. Mice exposed to binge drinking did not show an increase in cortical theta power and basal forebrain adenosine levels during sleep deprivation; NREM sleep and NREM delta power did not increase during recovery sleep suggesting that mice exposed to binge alcohol do not develop sleep pressure. Our final experiment examined expression of genes regulating sleep homeostasis following binge drinking. While binge drinking did not affect adenosine kinase and A1 receptor, expression of equilibrative nucleoside transporter 1 (ENT1) was significantly reduced. These results suggest that binge alcohol consumption-induced down-regulation of ENT1 expression may disrupt sleep homeostasis and cause sleep disturbances. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

Melatonin promotes sleep in mice by inhibiting orexin neurons in the perifornical lateral hypothalamus.

Melatonin promotes sleep. However, the underlying mechanisms are unknown. Orexin neurons in the perifornical lateral hypothalamus (PFH) are pivotal for wake promotion. Does melatonin promote sleep by inhibiting orexin neurons? We used C57BL/6J mice and designed 4 experiments to address this question. Experiment 1 used double-labeled immunofluorescence and examined the presence of melatonin receptors on orexin neurons. Second, mice, implanted with bilateral guides targeted toward PFH and sleep-recording electrodes, were infused with melatonin (500 pmole/50 nL/side) at dark onset (onset of active period), and spontaneous bouts of sleep-wakefulness were examined. Third, mice, implanted with bilateral guides into the PFH, were infused with melatonin (500 pmole/50 nL/side) at dark onset and euthanized 2 hours later, to examine the activation of orexin neurons using c-Fos expression in orexin neurons. Fourth, mice, implanted with PFH bilateral guides and sleep-recording electrodes, were infused with melatonin receptor antagonist, luzindole (10 pmol/50 nL/side), at light onset (onset of sleep period), and spontaneous bouts of sleep-wakefulness were examined. Our results suggest that orexin neurons express MT1, but not MT2 receptors. Melatonin infusion into the PFH, at dark onset, site-specifically and significantly increased NREM sleep (43.7%, P = .003) and reduced wakefulness (12.3%, P = .013). Local melatonin infusion at dark onset inhibited orexin neurons as evident by a significant reduction (66%, P = .0004) in the number of orexin neurons expressing c-Fos. Finally, luzindole infusion-induced blockade of melatonin receptors in PFH at sleep onset significantly increased wakefulness (44.1%, P = .015). Based on these results, we suggest that melatonin may act via the MT1 receptors to inhibit orexin neurons and promote sleep.

Sleep Medicine: Parasomnias.

Parasomnias are abnormal and undesirable behaviors during sleep and are thought to be due to the sleep state instability. Some of them are benign, while some of them point to a possible underlying neurodegenerative process. This article briefly discusses the clinical characteristics, demographics, and pathophysiology of major parasomnias and associated disorders. The classification outlined in this article conforms to the current version of International Classification of Sleep disorders.

Sleep Medicine: Restless Legs Syndrome.

Restless Legs Syndrome is a highly prevalent sensorimotor disorder characterized by urge to move the legs due to discomfort that primarily happens in the evening or at nights. Although the exact pathophysiology remains unclear, brain iron deficiency and altered dopaminergic function appears to play an important role in the pathogenesis of this condition. This disorder affects women more frequently and is associated with significant morbidity.

Lesion of the basal forebrain cholinergic neurons attenuates sleepiness and adenosine after alcohol consumption.

Alcohol has a profound effect on sleep. However, neuronal substrates mediating sleep-promoting effects of alcohol are unknown. Since the basal forebrain (BF) cholinergic neurons are implicated in the homeostatic regulation of sleep, we hypothesized that the BF cholinergic neurons may have an important role in sleepiness observed after alcohol consumption. 192-IgG-saporin (bilateral BF infusions) was used to selectively lesion BF cholinergic neurons in adult male Sprague-Dawley rats. Standard surgical procedures were used to implant sleep recording electrodes or microdialysis guide cannulas. The first experiment used between-group design [lesion and sham (controls)] and examined effects of BF cholinergic neuronal lesions on alcohol (3 g/Kg; ig) induced sleep promotion. The second experiment used within-group design [lesion (ipsilateral BF) and sham (controls; contralateral BF) in same animal] and local reverse microdialysis infusion of alcohol (300 mM) to examine the effects of cholinergic neuronal lesions on extracellular adenosine in the BF. Alcohol had a robust sleep promoting effect in controls as evidenced by a significant reduction in sleep onset latency and wakefulness; non-rapid eye movement sleep was significantly increased. No such alcohol-induced sleep promotion was observed in lesioned rats with significantly fewer BF cholinergic neurons. Rapid eye movement sleep was minimally affected. Adenosine release was significantly reduced following local infusion of alcohol on the lesion side, with significantly fewer cholinergic neurons as compared with the control side. Based on these results, we suggest that alcohol promotes sleep by increasing extracellular adenosine via its action on cholinergic neurons of the BF. Read the Editorial Highlight for this article on page 620.

Nicotine administration in the wake-promoting basal forebrain attenuates sleep-promoting effects of alcohol.

Nicotine and alcohol co-abuse is highly prevalent, although the underlying causes are unclear. It has been suggested that nicotine enhances pleasurable effects of alcohol while reducing aversive effects. Recently, we reported that nicotine acts via the basal forebrain (BF) to activate nucleus accumbens and increase alcohol consumption. Does nicotine suppress alcohol-induced aversive effects via the BF? We hypothesized that nicotine may act via the BF to suppress sleep-promoting effects of alcohol. To test this hypothesis, adult male Sprague-Dawley rats were implanted with sleep-recording electrodes and bilateral guides targeted toward the BF. Nicotine (75 pmol/500 nL/side) or artificial cerebrospinal fluid (ACSF; 500 nL/side) was microinjected into the BF followed by intragastric alcohol (ACSF + EtOH and NiC + EtOH groups; 3 g/kg) or water (NiC + W and ACSF + W groups; 10 mL/kg) administration. On completion, rats were killed and processed to localize injection sites in the BF. The statistical analysis revealed a significant effect of treatment on sleep-wakefulness. While rats exposed to alcohol (ACSF + EtOH) displayed strong sleep promotion, nicotine pre-treatment in the BF (NiC + EtOH) attenuated alcohol-induced sleep and normalized sleep-wakefulness. These results suggest that nicotine acts via the BF to suppress the aversive, sleep-promoting effects of alcohol, further supporting the role of BF in alcohol-nicotine co-use.

Nicotine administration in the cholinergic basal forebrain increases alcohol consumption in C57BL/6J mice.

BACKGROUND: Alcohol and nicotine are the most commonly abused drugs. The frequent co-morbidity of alcohol and nicotine addiction has led to the hypothesis that they may act via a common substrate: the nicotinic acetylcholine receptors (nAChRs) especially α4ß2 and α7 subtypes, the most prevalent nAChRs in the brain. Compelling evidence suggests that alcohol enhances the function of α4ß2 subtype. The FDA approved smoking cessation drug, varenicline ("Chantix"), a partial agonist of α4ß2 nAChR subtype, reduces alcohol self-administration and alcohol craving in humans and rodents. The cholinergic basal forebrain (BF) controls various functions including arousal, attention, and cognition, and there is a predominance of α4ß2 and α7 subtypes. We have shown that the BF has an important role in mediating the effects of alcohol and local infusion of nicotine in the BF activates nucleus accumbens. Does BF have any role in mediating the effect of nicotine on alcohol consumption? This study was designed to address this question. METHODS: Under standard surgical procedure, C57BL/6J mice were stereotaxically implanted with bilateral stainless steel guide cannula above the BF. Following post operative recovery and habituation, the animals were exposed to the "drinking-in-the-dark" paradigm whereby alcohol (20%) was presented for 2 hours daily for 3 days. On the fourth day, nicotine or artificial cerebrospinal fluid (ACSF) was microinjected bilaterally in the BF. After 1 hour, mice were exposed to alcohol and allowed to self-administer for 4 hours. The effect of BF nicotine infusion on sucrose consumption was also examined. On completion, mice were euthanized, brain removed and processed to localize the BF injection sites. RESULTS: As compared with the ACSF, bilateral nicotine injections into the BF significantly (p < 0.05; n = 5/group) increased alcohol consumption. Sucrose consumption remained unaffected. CONCLUSIONS: Based on our results, we believe that the BF may have an important role in nicotine-alcohol co-use.

Acute binge alcohol administration reverses sleep-wake cycle in Sprague Dawley rats.

BACKGROUND: Binge alcohol drinking is among the most common pattern of alcohol consumption in our society. Binge alcohol consumption has serious negative consequence on mental and physical health. Although alcohol consumption is known to have profound impact on sleep, it is yet unknown as to how binge alcohol affects/alters sleep-wakefulness. The objective of this study was to examine the effect of acute binge alcohol administration on sleep-wakefulness. METHODS: Male Sprague Dawley rats were used in the study. Under standard aseptic surgical conditions, rats (N = 7) were implanted with sleep-recording electrodes. After postoperative recovery and habituation, baseline sleep-wakefulness was recorded. Subsequently, rats were exposed to binge alcohol treatment as follows: One hour before light onset, a priming dose of 5 g/kg of alcohol was administered followed by 2 subsequent doses (adjusted based on the intoxication level of the rat) approximately 8 hours apart. Sleep-wakefulness was continuously recorded for 3 days post-binge. RESULTS: Acute binge alcohol administration had no significant effect on sleep-wakefulness on post-binge Day 1. However, on post-binge Day 2, after blood alcohol concentration (BAC) was 0, sleep disruptions were observed manifested by a reversal of sleep-wakefulness as evident from insomnia-like symptoms (significant increase in wakefulness; significant reduction in nonrapid eye movement [NREM] sleep) during the normal sleep (light) period and excessive sleep (significant increase in NREM sleep) during the normal active (dark) period similar to excessive daytime sleepiness in humans. All sleep-wakefulness changes were normalized on Day 3 post-binge. CONCLUSIONS: Alcohol hangover is defined as the presence of unpleasant symptoms that peak when BAC is 0. Our results suggest that the reversal of sleep-wakefulness accompanies alcohol hangover after binge alcohol administration.

Nicotine infusion in the wake-promoting basal forebrain enhances alcohol-induced activation of nucleus accumbens.

BACKGROUND: Nicotine and alcohol co-abuse is highly prevalent. Recently, we have shown that nicotine infusion in the basal forebrain (BF) increases alcohol consumption. As nucleus accumbens (NAc) is the terminal brain region associated with drug addiction, we hypothesize that nicotine infusion in the BF may enhance alcohol-induced activation of NAc. METHODS: Adult male Sprague-Dawley rats were surgically implanted with bilateral guide cannulas in the BF. Following postoperative recovery, rats were divided into 4 groups: (i) ACSF + W group received artificial cerebrospinal fluid (ACSF; 500 nl/side) in the BF and systemic water (intragastric [ig]; 10 ml/kg; N = 5), (ii) ethanol (EtOH) group received ACSF in the BF (500 nl/side) and systemic alcohol (ig; 3 g/kg; N = 5), (iii) NiC group received nicotine in the BF (75 pmole/500 nl/side) and systemic water (ig; 10 ml/kg; N = 5), and (iv) NiC + EtOH group received nicotine in the BF (75 pmole/500 nl/side) and systemic alcohol (ig; 3 g/kg; N = 5). Rats were euthanized 2 hours after treatment to examine c-Fos expression in the NAc by immunohistochemistry. RESULTS: All injections sites were localized in the BF. Two-way analysis of variance (ig vs. infusion) revealed significant main effects of both treatments (ig and infusion, p < 0.001) on c-Fos expression in the NAc shell, but not in the core. Subsequent post hoc test (Bonferroni's) revealed that as compared to ACSF + W group, c-Fos expression was significantly increased in the shell of NAc of rats in all 3 (EtOH, NiC, and NiC + EtOH) groups with maximal increase observed in NiC + EtOH group. CONCLUSIONS: The results suggest the following: (i) BF nicotine infusion induced c-Fos in both core and the shell region of NAc at levels comparable to those observed after systemic alcohol administration; (ii) BF nicotine infusion with systemic alcohol induced a significant additive increase in c-Fos expression only in the NAc shell region. These findings implicate the BF in alcohol and nicotine co-use.

Adenosine and glutamate signaling in neuron-glial interactions: implications in alcoholism and sleep disorders.

Recent studies have demonstrated that the function of glia is not restricted to the support of neuronal function. Especially, astrocytes are essential for neuronal activity in the brain. Astrocytes actively participate in synapse formation and brain information processing by releasing or uptaking gliotransmitters such as glutamate, d-serine, adenosine 5'-triphosphate (ATP), and adenosine. In the central nervous system, adenosine plays an important role in regulating neuronal activity as well as in controlling other neurotransmitter systems such as GABA, glutamate, and dopamine. Ethanol (EtOH) increases extracellular adenosine levels, which regulates the ataxic and hypnotic/sedative (somnogenic) effects of EtOH. Adenosine signaling is also involved in the homeostasis of major inhibitory/excitatory neurotransmission (i.e., GABA or glutamate) through neuron-glial interactions, which regulates the effect of EtOH and sleep. Adenosine transporters or astrocytic SNARE-mediated transmitter release regulates extracellular or synaptic adenosine levels. Adenosine then exerts its function through several adenosine receptors and regulates glutamate levels in the brain. This review presents novel findings on how neuron-glial interactions, particularly adenosinergic signaling and glutamate uptake activity involving glutamate transporter 1 (GLT1), are implicated in alcoholism and sleep disorders.

Ischemic Stroke Disrupts Sleep Homeostasis in Middle-Aged Mice.

Sleep disturbances, including insomnia and excessive daytime sleepiness, are highly prevalent in patients with ischemic stroke (IS), which severely impacts recovery and rehabilitation efforts. However, how IS induces sleep disturbances is unclear. Three experiments were performed on middle-aged C57BL/6J mice, instrumented with sleep recording electrodes and/or subjected to 1 h of middle cerebral artery (MCAO; Stroke group) or sham (Sham group) occlusion to induce IS. After 48 h of reperfusion (a) experiment 1 verified sensorimotor deficit (using Garcia scale) and infarction (using TTC staining) in this mouse model; (b) experiment 2 examined the effects of IS on the quality (sleep latency and NREM delta power) and quantity (duration) of sleep; and (c) experiment 3 determined the effects of IS on sleep homeostasis using sleep deprivation (SD) and recovery sleep (RS) paradigm. Stroke mice display (a) a significant correlation between sensorimotor deficit and cerebral infarction; (b) insomnia-like symptoms (increased sleep latency, reduced NREM duration and delta power) during the light (inactive) period and daytime sleepiness-like symptoms during the dark (active) period mimicking sleep in IS patients; and (c) impairments in the markers of sleep pressure (during SD) and sleep dissipation (during RS). Our results suggest that IS disrupts sleep homeostasis to cause sleep disturbances.

Implication of the purinergic system in alcohol use disorders.

In the central nervous system, adenosine and adenosine 5'-triphosphate (ATP) play an important role in regulating neuronal activity as well as controlling other neurotransmitter systems, such as, GABA, glutamate, and dopamine. Ethanol increases extracellular adenosine levels that regulate the ataxic and hypnotic/sedative effects of ethanol. Interestingly, ethanol is known to increase adenosine levels by inhibiting an ethanol-sensitive adenosine transporter, equilibrative nucleoside transporter type 1 (ENT1). Ethanol is also known to inhibit ATP-specific P2X receptors, which might result in such similar effects as those caused by an increase in adenosine. Adenosine and ATP exert their functions through P1 (metabotropic) and P2 (P2X-ionotropic and P2Y-metabotropic) receptors, respectively. Purinergic signaling in cortex-striatum-ventral tegmental area (VTA) has been implicated in regulating cortical glutamate signaling as well as VTA dopaminergic signaling, which regulates the motivational effect of ethanol. Moreover, several nucleoside transporters and receptors have been identified in astrocytes, which regulate not only adenosine-ATP neurotransmission, but also homeostasis of major inhibitory-excitatory neurotransmission (i.e., GABA or glutamate) through neuron-glial interactions. This review will present novel findings on the implications of adenosine and ATP neurotransmission in alcohol use disorders.

Short-term sleep deprivation immediately after contextual conditioning inhibits BDNF signaling and disrupts memory consolidation in predator odor trauma mice model of PTSD.

Post-traumatic stress disorder (PTSD) is a debilitating neuropsychiatric illness affecting > 7 million people every year in the US. Recently, we have shown that the mouse model of predator odor trauma (POT) displayed contextual conditioning and core features of PTSD including sleep disturbances (hyperarousal) and retrieval of traumatic memories following exposure to objective reminders (re-experiencing). PTSD is a disorder of memory function. Since memory consolidation requires the expression of BDNF along with an activation of MAPK/pERK signaling pathway in limbic brain structures (hippocampus and amygdala) and sleep favors memory consolidation, we hypothesized that short-term sleep deprivation (SD, 3 h), immediately after contextual conditioning will attenuate molecular correlates of memory consolidation, sleep disturbances, and memory consolidation. We performed two experiments in adult male C57BL/6J mice to test our hypothesis. Experiment 1 determined the effects of SD on contextual conditioning and changes in sleep wakefulness. Experiment 2 determined the effects of SD on contextual conditioning-induced changes in the expression of BDNF and pERK in hippocampus and amygdala. SD immediately after contextual conditioning (POT + SD group) significantly attenuated sleep disturbances, memory retrieval, and expression of pERK and BDNF in the hippocampus and amygdala as compared to POT-SD group (no SD after contextual conditioning). No significant differences were observed between POT + SD, NOC-SD (no contextual conditioning + no SD), and NOC + SD (no contextual conditioning + SD) groups. Memory consolidation requires sleep and the expression of pERK and BDNF in hippocampus and amygdala immediately after contextual conditioning in POT model of PTSD in mice.

Rats exposed to chronic alcohol display protracted insomnia and daytime sleepiness-like behavior during alcohol withdrawal✰.

INTRODUCTION: Alcohol use disorder (AUD), a chronic brain disorder, is characterized by a multitude of symptoms, including insomnia, during withdrawal. Previously, we have shown that rats exposed to chronic alcohol displayed insomnia-like symptoms during acute withdrawal. Since insomnia lasts for several years and is a major risk factor of relapse to alcoholism, the present study is designed to investigate the long-term effects of alcohol withdrawal on sleep-wakefulness. METHODS: Adult male Sprague-Dawley rats, instrumented with sleep recording electrodes, were divided into two groups: Alcohol and Control. Rats were either administered alcohol (35% v/v), mixed with infant formula (Alcohol group) or control mixture containing water and infant formula (Controls; 10 mL/kg) every 8 h for 4 days using Majchrowicz's chronic binge drinking protocol. Electrographic recordings of sleep-wakefulness were performed until withdrawal day 7, however, the data was analyzed for withdrawal days 3, 5 and 7 in both Control and Alcohol groups. RESULTS: As compared to the controls, alcohol-exposed rats displayed insomnia-like symptoms as revealed by a) significant reduction in the quantity and quality of sleep during the light (inactive) period and b) a significant increase in NREM sleep with a concomitant reduction in the amount of time spent in the wakefulness during the dark (active) period of alcohol withdrawal. CONCLUSION: Our results suggest that the chronic binge model of alcohol dependence mimics clinical symptoms of AUD especially protracted insomnia and is suitable for understanding the mechanisms associated with alcohol withdrawal-induced behaviors.

Multi-focus Image Fusion for Confocal Microscopy Using U-Net Regression Map.

Characterizing the spatial relationship between blood vessel and lymphatic vascular structures, in the mice dura mater tissue, is useful for modeling fluid flows and changes in dynamics in various disease processes. We propose a new deep learning-based approach to fuse a set of multi-channel single-focus microscopy images within each volumetric z-stack into a single fused image that accurately captures as much of the vascular structures as possible. The red spectral channel captures small blood vessels and the green fluorescence channel images lymphatics structures in the intact dura mater attached to bone. The deep architecture Multi-Channel Fusion U-Net (MCFU-Net) combines multi-slice regression likelihood maps of thin linear structures using max pooling for each channel independently to estimate a slice-based focus selection map. We compare MCFU-Net with a widely used derivative-based multi-scale Hessian fusion method [8]. The multi-scale Hessian-based fusion produces dark-halos, non-homogeneous backgrounds and less detailed anatomical structures. Perception based no-reference image quality assessment metrics PIQUE, NIQE, and BRISQUE confirm the effectiveness of the proposed method.

Role of adenosine and wake-promoting basal forebrain in insomnia and associated sleep disruptions caused by ethanol dependence.

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.

Knockdown of orexin type 1 receptor in rat locus coeruleus increases REM sleep during the dark period.

The locus coeruleus (LC) regulates sleep/wakefulness and is densely innervated by orexinergic neurons in the lateral hypothalamus. Here we used small interfering RNAs (siRNAs) to test the role of LC orexin type 1 receptor (OxR1) in sleep­wake control. In sleep studies, bilateral OxR1 siRNA injections led to an increase of time spent in rapid eye movement (REM) sleep, which was selective for the dark (active) period, peaked at approximately 30% of control during the second dark period after injection and then disappeared after 4 days. Cataplexy-like episodes were not observed. The percentage time spent in wakefulness and non-REM (NREM) sleep and the power spectral profile of NREM and REM sleep were unaffected. Control animals, injected with scrambled siRNA, had no sleep changes after injection. Quantification of the knockdown revealed that unilateral microinjection of siRNAs targeting OxR1 into the rat LC on two consecutive days induced a 45.5% reduction of OxR1 mRNA in the LC 2 days following the injections when compared with the contralateral side receiving injections of control (scrambled) siRNAs. This reduction disappeared 4 days after injection. Similarly, unilateral injection of OxR1 siRNA into the LC revealed a marked (33.5%) reduction of OxR1 staining 2 days following injections. In contrast, both the mRNA level and immunohistochemical staining for tyrosine hydroxylase were unaffected. The results indicate that a modest knockdown of OxR1 is sufficient to induce observable sleep changes. Moreover, orexin neurons, by acting on OxR1 in the LC, play a role in the diurnal gating of REM sleep.

Effects of ethanol on extracellular levels of adenosine in the basal forebrain: an in vivo microdialysis study in freely behaving rats.

BACKGROUND: Adenosine is implicated to play a pivotal role in mediating many neuronal responses to ethanol. While in vitro studies performed in cell culture have demonstrated that acute ethanol exposure increases extracellular adenosine levels, this effect has not been demonstrated, in vivo, in the brain. We performed an in vivo microdialysis study to examine the effects of local ethanol perfusion on extracellular levels of adenosine in the basal forebrain (BF). METHODS: Under sterile conditions and using a standard surgical protocol, adult male Sprague-Dawley rats were implanted with unilateral microdialysis guide cannula targeted toward the BF. Following postoperative recovery, the microdialysis probe was inserted. After allowing at least 12 to 16 hours for probe insertion recovery, the experiment was begun. Artificial cerebrospinal fluid (aCSF) was perfused (0.7 microl/min) for 80 minutes, and 4 x 20-minute pre-ethanol baseline samples were collected. Subsequently, 30, 100, and 300 mM doses of ethanol were perfused. Each ethanol dose was perfused for 80 minutes, and 4 x 20-minute samples were collected. Finally, aCSF was perfused, and 4 x 20 postethanol samples were collected. Adenosine in the microdialysate was separated and measured with HPLC coupled with an UV detector. On completion, the animals were euthanized, brain removed and processed for histology. RESULTS: Local ethanol perfusion in the BF produced a significant increase in extracellular adenosine with the highest dose of 300 mM ethanol producing a 4-fold increase. Cresyl violet (Nissl) staining did not indicate any toxic damage in the area surrounding the probe tip. Choline acetyltransferase immunohistochemistry revealed that all microdialysis probe sites were localized in the BF. CONCLUSION: Our study is the first to demonstrate that ethanol acts directly in the brain to increase extracellular adenosine.

Role of wake-promoting basal forebrain and adenosinergic mechanisms in sleep-promoting effects of ethanol.

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 3 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 the first 12 hours postethanol. 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.

Chronic alcohol exposure reduces acetylated histones in the sleep-wake regulatory brain regions to cause insomnia during withdrawal.

BACKGROUND: Alcohol use disorder (AUD) develops after chronic and heavy use of alcohol. Insomnia, a hallmark of AUD, plays a crucial role in the development of AUD. However, the causal mechanisms are unknown. Since chronic alcohol reduces acetylated histones and disrupts the epigenome, we hypothesized that chronic alcohol exposure will reduce acetylated histones in wake-promoting regions of the brain to cause insomnia during alcohol withdrawal. METHODS: Adult male C57BL/6J mice, surgically instrumented for electrophysiological monitoring of sleep-wakefulness, were exposed to chronic alcohol (6.8%) consumption using Lieber-DeCarli liquid diet. Three experiments were performed. First, the effect of chronic alcohol consumption was examined on sleep-wakefulness during 7 days of withdrawal. Second, the expression of acetylated histones, H3 lysine 14 (AcH3K14), was examined in two major sleep-wake regulatory brain regions: basal forebrain (BF) and lateral hypothalamus (LH) of the brain by using western blotting. Next, blockade of histone deacetylase, via systemic administration of TSA was examined on alcohol-induced changes in sleep-wakefulness. RESULTS: Alcoholic mice displayed a significant reduction in the quality and quantity of NREM sleep coupled with a significant increase in wakefulness that lasted for several days during alcohol withdrawal. In addition, alcoholic mice displayed a significant reduction in the expression of AcH3K14 in both BF and LH. Systemic administration of TSA significantly attenuated insomnia and improved the quality and quantity of sleep during alcohol withdrawal. CONCLUSIONS: Based on our results, we suggest that a causal relationship exists between reduced histone acetylation and insomnia during alcohol withdrawal.