Christina L. Ruby

Chronic Ethanol Disrupts Circadian Photic Entrainment and Daily Locomotor Activity in the Mouse

BACKGROUND Chronic ethanol abuse is associated with disrupted circadian rhythms and sleep. Ethanol administration impairs circadian clock phase-resetting, suggesting a mode for the disruptive effect of alcohol abuse on circadian timing. Here, we extend previous studies to explore the effects of chronic forced ethanol on photic phase-resetting, photic entrainment, and daily locomotor activity patterns in C57BL/6J mice. METHODS First, microdialysis was used to characterize the circadian patterns of ethanol uptake in the suprachiasmatic (SCN) circadian clock and correlate this with systemic ethanol levels and episodic drinking of 10 or 15% ethanol. Second, the effects of chronic forced ethanol drinking and withdrawal on photic phase-delays of the circadian activity rhythm were assessed. Third, the effects of chronic ethanol drinking on entrainment to a weak photic zeitgeber (1 minute of 25 lux intensity light per day) were assessed. This method was used to minimize any masking actions of light that could mask ethanol effects on clock entrainment. RESULTS Peak ethanol levels in the SCN and periphery occurred during the dark phase and coincided with the time when light normally induces phase-delays in mice. These delays were dose-dependently inhibited by chronic ethanol and its withdrawal. Chronic ethanol did not impede re-entrainment to a shifted light cycle but affected entrainment under the weak photic zeitgeber and disrupted the daily pattern of locomotor activity. CONCLUSIONS These results confirm that chronic ethanol consumption and withdrawal markedly impair circadian clock photic phase-resetting. Ethanol also disturbs the temporal structure of nighttime locomotor activity and photic entrainment. Collectively, these results suggest a direct action of ethanol on the SCN clock.

Adenosine Transporter ENT1 Regulates the Acquisition of Goal-Directed Behavior and Ethanol Drinking through A2A Receptor in the Dorsomedial Striatum

Adenosine signaling has been implicated in the pathophysiology of many psychiatric disorders including alcoholism. Striatal adenosine A2A receptors (A2AR) play an essential role in both ethanol drinking and the shift from goal-directed action to habitual behavior. However, direct evidence for a role of striatal A2AR signaling in ethanol drinking and habit development has not been established. In the present study, we found that decreased A2AR-mediated CREB activity in the dorsomedial striatum (DMS) enhanced initial behavioral acquisition of goal-directed behaviors and the vulnerability to progress to excessive ethanol drinking during operant conditioning in mice lacking ethanol-sensitive adenosine transporter ENT1 (ENT1−/−). Using mice expressing β-galactosidase (lacZ) under the control of seven repeated CRE sites in both genotypes (CRE-lacZ/ENT1+/+ mice and CRE-lacZ/ENT1−/− mice) and the dominant-negative form of CREB, we found that reduced CREB activity in the DMS was causally associated with decreased A2AR signaling and increased goal-directed ethanol drinking. Finally, we have demonstrated that the A2AR antagonist ZM241385 dampened protein kinase A activity–mediated signaling in the DMS and promoted excessive ethanol drinking in ENT1+/+ mice, but not in ENT1−/− mice. Our results indicate that A2AR-mediated CREB signaling in the DMS is a key determinant in enhancing the development of goal-directed ethanol drinking in mice.

Chronic ethanol attenuates circadian photic phase resetting and alters nocturnal activity patterns in the hamster

Acute ethanol (EtOH) administration impairs circadian clock phase resetting, suggesting a mode for the disruptive effect of alcohol abuse on human circadian rhythms. Here, we extend this research by characterizing the chronobiological effects of chronic alcohol consumption. First, daily profiles of EtOH were measured in the suprachiasmatic nucleus (SCN) and subcutaneously using microdialysis in hamsters drinking EtOH. In both cases, EtOH peaked near lights-off and declined throughout the dark-phase to low day-time levels. Drinking bouts preceded EtOH peaks by approximately 20 min. Second, hamsters chronically drinking EtOH received a light pulse during the late dark phase [Zeitgeber time (ZT) 18.5] to induce photic phase advances. Water controls had shifts of 1.2 +/- 0.2 h, whereas those drinking 10% and 20% EtOH had much reduced shifts (0.5 +/- 0.1 and 0.3 +/- 0.1 h, respectively; P < 0.001 vs. controls). Third, incremental decreases in light intensity (270 lux to 0.5 lux) were used to explore chronic EtOH effects on photic entrainment and rhythm stability. Activity onset was unaffected by 20% EtOH at all light intensities. Conversely, the 24-h pattern of activity bouts was disrupted by EtOH under all light intensities. Finally, replacement of chronic EtOH with water was used to examine withdrawal effects. Water controls had photic phase advances of 1.1 +/- 0.3 h, while hamsters deprived of EtOH for 2-3 days showed enhanced shifts (2.1 +/- 0.3 h; P < 0.05 vs. controls). Thus, in chronically drinking hamsters, brain EtOH levels are sufficient to inhibit photic phase resetting and disrupt circadian activity. Chronic EtOH did not impair photic entrainment; however, its replacement with water potentiated photic phase resetting.

Acute ethanol impairs photic and nonphotic circadian phase resetting in the Syrian hamster

Disrupted circadian rhythmicity is associated with ethanol (EtOH) abuse, yet little is known about how EtOH affects the mammalian circadian clock of the suprachiasmatic nucleus (SCN). Clock timing is regulated by photic and nonphotic inputs to the SCN involving glutamate release from the retinohypothalamic tract and serotonin (5-HT) from the midbrain raphe, respectively. Our recent in vitro studies in the SCN slice revealed that EtOH blocks photic phase-resetting action of glutamate and enhances the nonphotic phase-resetting action of the 5-HT1A,7 agonist, 8-OH-DPAT. To explore the basis of these effects in the whole animal, we used microdialysis to characterize the pharmacokinetics of intraperitoneal injection of EtOH in the hamster SCN extracellular fluid compartment and then studied the effects of such EtOH treatment on photic and serotonergic phase resetting of the circadian locomotor activity rhythm. Peak EtOH levels (approximately 50 mM) from a 2 g/kg injection occurred within 20-40 min with a half-life of approximately 3 h. EtOH treatment dose-dependently attenuated photic phase advances but had no effect on phase delays and, contrary to in vitro findings, markedly attenuated 8-OH-DPAT-induced phase advances. In a complementary experiment using reverse microdialysis to deliver a timed SCN perfusion of EtOH during a phase-advancing light pulse, the phase advances were blocked, similar to systemic EtOH treatment. These results are evidence that acute EtOH significantly affects photic and nonphotic phase-resetting responses critical to circadian clock regulation. Notably, EtOH inhibition of photic signaling is manifest through direct action in the SCN. Such actions could underlie the disruption of circadian rhythmicity associated with alcohol abuse.

Striatal Adenosine Signaling Regulates EAAT2 and Astrocytic AQP4 Expression and Alcohol Drinking in Mice

Adenosine signaling is implicated in several neuropsychiatric disorders, including alcoholism. Among its diverse functions in the brain, adenosine regulates glutamate release and has an essential role in ethanol sensitivity and preference. However, the molecular mechanisms underlying adenosine-mediated glutamate signaling in neuroglial interaction remain elusive. We have previously shown that mice lacking the ethanol-sensitive adenosine transporter, type 1 equilibrative nucleoside transporter (ENT1), drink more ethanol compared with wild-type mice and have elevated striatal glutamate levels. In addition, ENT1 inhibition or knockdown reduces glutamate transporter expression in cultured astrocytes. Here, we examined how adenosine signaling in astrocytes contributes to ethanol drinking. Inhibition or deletion of ENT1 reduced the expression of type 2 excitatory amino-acid transporter (EAAT2) and the astrocyte-specific water channel, aquaporin 4 (AQP4). EAAT2 and AQP4 colocalization was also reduced in the striatum of ENT1 null mice. Ceftriaxone, an antibiotic compound known to increase EAAT2 expression and function, elevated not only EAAT2 but also AQP4 expression in the striatum. Furthermore, ceftriaxone reduced ethanol drinking, suggesting that ENT1-mediated downregulation of EAAT2 and AQP4 expression contributes to excessive ethanol consumption in our mouse model. Overall, our findings indicate that adenosine signaling regulates EAAT2 and astrocytic AQP4 expressions, which control ethanol drinking in mice.

Attenuation of Ethanol Withdrawal by Ceftriaxone-Induced Upregulation of Glutamate Transporter EAAT2

Alcohol withdrawal syndrome (AWS) is a potentially fatal outcome of severe alcohol dependence that presents a significant challenge to treatment. Although AWS is thought to be driven by a hyperglutamatergic brain state, benzodiazepines, which target the GABAergic system, comprise the first line of treatment for AWS. Using a rat model of ethanol withdrawal, we tested whether ceftriaxone, a β-lactam antibiotic known to increase the expression and activity of glutamate uptake transporter EAAT2, reduces the occurrence or severity of ethanol withdrawal manifestations. After a 2-week period of habituation to ethanol in two-bottle choice, alcohol-preferring (P) and Wistar rats received ethanol (4.0 g/kg) every 6 h for 3–5 consecutive days via gavage. Rats were then deprived of ethanol for 48 h during which time they received ceftriaxone (50 or 100 mg/kg, IP) or saline twice a day starting 12 h after the last ethanol administration. Withdrawal manifestations were captured by continuous video recording and coded. The evolution of ethanol withdrawal was markedly different for P rats vs Wistar rats, with withdrawal manifestations occurring >12 h later in P rats than in Wistar rats. Ceftriaxone 100 mg/kg per injection twice per day (200 mg/kg/day) reduced or abolished all manifestations of ethanol withdrawal in both rat variants and prevented withdrawal-induced escalation of alcohol intake. Finally, ceftriaxone treatment was associated with lasting upregulation of ethanol withdrawal-induced downregulation of EAAT2 in the striatum. Our data support the role of ceftriaxone in alleviating alcohol withdrawal and open a novel pharmacologic avenue that requires clinical evaluation in patients with AWS.

An Essential Role for Adenosine Signaling in Alcohol Abuse

In the central nervous system (CNS), adenosine plays an important role in regulating neuronal activity and modulates signaling by other neurotransmitters, including GABA, glutamate, and dopamine. Adenosine suppresses neurotransmitter release, reduces neuronal excitability, and regulates ion channel function through activation of four classes of G protein-coupled receptors, A(1), A(2A), A(2B), and A(3). Central adenosine are largely controlled by nucleoside transporters, which transport adenosine levels across the plasma membrane. Adenosine has been shown to modulate cortical glutamate signaling and ventral-tegmental dopaminergic signaling, which are involved in several aspects of alcohol use disorders. Acute ethanol elevates extracellular adenosine levels by selectively inhibiting the type 1 equilibrative nucleoside transporter, ENT1. Raised adenosine levels mediate the ataxic and sedative/hypnotic effects of ethanol through activation of A(1) receptors in the cerebellum, striatum, and cerebral cortex. Recently, we have shown that pharmacological inhibition or genetic deletion of ENT1 reduces the expression of excitatory amino acid transporter 2 (EAAT2), the primary regulator of extracellular glutamate, in astrocytes. These lines of evidence support a central role for adenosine-mediated glutamate signaling and the involvement of astrocytes in regulating ethanol intoxication and preference. In this paper, we discuss recent findings on the implication of adenosine signaling in alcohol use disorders.

Acute ethanol disrupts photic and serotonergic circadian clock phase-resetting in the mouse.

BACKGROUND Alcohol dependence is associated with impaired circadian rhythms and sleep. Ethanol administration disrupts circadian clock phase-resetting, suggesting a mode for the disruptive effect of alcohol dependence on the circadian timing system. In this study, we extend previous work in C57BL/6J mice to: (i) characterize the suprachiasmatic nucleus (SCN) pharmacokinetics of acute systemic ethanol administration, (ii) explore the effects of acute ethanol on photic and nonphotic phase-resetting, and (iii) determine if the SCN is a direct target for photic effects. METHODS First, microdialysis was used to characterize the pharmacokinetics of acute intraperitoneal (i.p.) injections of 3 doses of ethanol (0.5, 1.0, and 2.0 g/kg) in the mouse SCN circadian clock. Second, the effects of acute i.p. ethanol administration on photic phase delays and serotonergic ([+]8-OH-DPAT-induced) phase advances of the circadian activity rhythm were assessed. Third, the effects of reverse-microdialysis ethanol perfusion of the SCN on photic phase-resetting were characterized. RESULTS Peak ethanol levels from the 3 doses of ethanol in the SCN occurred within 20 to 40 minutes postinjection with half-lives for clearance ranging from 0.6 to 1.8 hours. Systemic ethanol treatment dose-dependently attenuated photic and serotonergic phase-resetting. This treatment also did not affect basal SCN neuronal activity as assessed by Fos expression. Intra-SCN perfusion with ethanol markedly reduced photic phase delays. CONCLUSIONS These results confirm that acute ethanol attenuates photic phase-delay shifts and serotonergic phase-advance shifts in the mouse. This dual effect could disrupt photic and nonphotic entrainment mechanisms governing circadian clock timing. It is also significant that the SCN clock is a direct target for disruptive effects of ethanol on photic shifting. Such actions by ethanol could underlie the disruptive effects of alcohol abuse on behavioral, physiological, and endocrine rhythms associated with alcoholism.

ENVIRONMENTAL MODULATION OF ALCOHOL INTAKE IN HAMSTERS: EFFECTS OF WHEEL-RUNNING AND CONSTANT LIGHT EXPOSURE

BACKGROUND Alcohol abuse leads to marked disruptions of circadian rhythms, and these disturbances in themselves can increase the drive to drink. Circadian clock timing is regulated by light, as well as by nonphotic influences such as food, social interactions, and wheel running. We previously reported that alcohol markedly disrupts photic and nonphotic modes of circadian rhythm regulation in Syrian hamsters. As an extension of this work, we characterize the hedonic interrelationship between wheel running and ethanol (EtOH) intake and the effects of environmental circadian disruption (long-term exposure to constant light [LL]) on the drive to drink. METHODS First, we tested the effect of wheel running on chronic free-choice consumption of a 20% (v/v) EtOH solution and water. Second, the effect of this alcohol drinking on wheel running in alcohol-naive animals was investigated. Third, we assessed the influence of LL, known to suppress locomotor activity and cause circadian rhythm disruption, on EtOH consumption and wheel-running behavior. RESULTS Inhibitory effects of wheel running on EtOH intake and vice versa were observed. Exposure to LL, while not affecting EtOH intake, induced rhythm splitting in 75% of the animals. Notably, the splitting phenotype was associated with lower levels of EtOH consumption and preference prior to, and throughout, the period of LL exposure. CONCLUSIONS These results are evidence that exercise may offer an efficacious clinical approach to reducing EtOH intake. Also, predisposition for light-induced (or other) forms of circadian disruption may modulate the drive to drink.

Adenosinergic Regulation of Striatal Clock Gene Expression and Ethanol Intake During Constant Light

Circadian rhythm and sleep disruptions occur frequently in individuals with alcohol use disorders (AUD) and present significant barriers to treatment. Recently, a variant of adenosine transporter, equilibrative nucleoside transporter 1 (ENT1), was associated with the co-occurrence of sleep problems and AUD. We have previously shown that mice lacking ENT1 (ENT1 KO) have reduced adenosine levels in the striatum and drink more alcohol compared with wild types (WT). However, it is unknown whether ENT1 deletion disrupts circadian rhythms, which may contribute to alcohol preference in ENT1 KO mice. Here we used these mice to determine whether endogenous adenosine regulates circadian genetic and behavioral rhythms and influences alcohol intake during chronodisruption. We examined circadian locomotor activity in ENT1 KO vs WT littermates and found that ENT1 KO mice were both active earlier and hyperactive compared with WT mice at night. We used real-time PCR and immunohistochemistry to estimate striatal clock gene levels and found that PER2 expression in the striatum was blunted by ENT1 deletion or A2A receptor (A2AR) antagonism. Next, we exposed ENT1 KO and WT mice to constant light (LL) and found further elevation in ethanol intake in ENT1 KO, but not in WT mice, supporting the notion that circadian dysfunction may contribute to increased alcohol intake in ENT1 KO mice. Finally, we showed that A2AR agonist administration normalized PER1 and PER2 expression and circadian locomotor activity in ENT1 KO mice. Together, our results demonstrate that adenosine signaling regulates cellular and behavioral circadian timing and influences alcohol intake during chronodisruption.