Long-term Ethanol Self-administration Research Articles

Changes in glucocorticoid receptor sensitivity following long‐term ethanol self‐administration in male and female rhesus macaques

Stress can alter ethanol consumption and long-term use results in adaptation of the stress response, highlighting a bidirectional relationship that remains poorly understood. Non-human primates are an excellent model to investigate the interaction of stress and long-term ethanol use due to their similar neuroendocrine system to humans and propensity to chronically drink intoxicating amounts of alcohol. Here, circulating stress hormones (adrenocorticotropin hormone (ACTH) and cortisol) were measured in response to a low (17 mg/kg) and high (130 mg/kg) dose of dexamethasone, a potent glucocorticoid receptor (GR) agonist. The dexamethasone response was compared before (baseline) and after 7 months of daily 22 h/d ethanol self-administration in young adult male (n=6) and female (n=6) rhesus macaques. At both timepoints, the high dose dexamethasone challenge resulted in 90-100% suppression of both ACTH and cortisol with no difference between sexes. At baseline, low dose dexamethasone reduced both ACTH and cortisol, but to a lesser degree and with greater between-subject variability, 0 – 60%. Following seven-months of daily ethanol consumption (average intakes ranging from 0.6 – 4.0 g/kg/day) the response to low dose dexamethasone was greater. This effect was more robust in females compared to males. Additionally, the change in suppression between baseline and following long-term daily ethanol consumption correlated with daily intake, such that the magnitude of change over time was greater in heavier drinkers. Because stress increases reliance on putamen-dependent or habitual behavior and heavy ethanol drinking leads to excitatory-inhibitory neurotransmission imbalance within the putamen, we tested the hypothesis that long-term ethanol self-administration will alter striatal transmission via the GR. Therefore, we used whole cell patch clamp electrophysiology to investigate the effect of bath application of dexamethasone onto putamen medium spiny neurons (MSNs). Dexamethasone rapidly decreased the area and amplitude of spontaneous inhibitory postsynaptic currents among binge drinkers. These data show that acute dexamethasone, presumably via activation of the membrane bound GR, activated fewer GABAergic receptors among binge drinkers. These data highlight a unique role of the GR within the putamen that may mediate stress dependent changes contributing to addiction vulnerability, particularly among binge drinkers.

Synaptic adaptations in the central amygdala and hypothalamic paraventricular nucleus associated with protracted ethanol abstinence in male rhesus monkeys.

Alcohol use disorder is a significant global burden. Stress has been identified as an etiological factor in the initiation and continuation of ethanol consumption. Understanding adaptations within stress circuitry is an important step toward novel treatment strategies. The effects of protracted abstinence following long-term ethanol self-administration on the central nucleus of the amygdala (CeA) and the hypothalamic paraventricular nucleus (PVN) were evaluated in male rhesus monkeys. Using whole-cell patch-clamp electrophysiology, inhibitory GABAergic transmission in the CeA and excitatory glutamatergic transmission in the PVN were measured. CeA neurons from abstinent drinkers displayed an elevated baseline spontaneous inhibitory postsynaptic current (sIPSC) frequency compared with controls, indicating increased presynaptic GABA release. Application of acute ethanol significantly increased the frequency of sIPSCs in controls, but not in abstinent drinkers, suggesting a tolerance to ethanol-enhanced GABA release in abstinent rhesus monkeys with a history of chronic ethanol self-administration and repeated abstinence. In the PVN, the frequency of spontaneous excitatory postsynaptic currents (sEPSC) was elevated in abstinent drinkers compared with controls, indicating increased presynaptic glutamate release. Notably, acute ethanol decreased presynaptic glutamate release onto parvocellular PVN neurons in both controls and abstinent drinkers, suggesting a lack of tolerance to acute ethanol among PVN neurons. These results are the first to demonstrate distinct synaptic adaptations and ethanol sensitivity in both the extrahypothalamic and hypothalamic stress circuits in abstinent rhesus males. Importantly, our findings describe adaptations in stress circuitry present in the brain at a state during abstinence, just prior to relapse to ethanol drinking.

Long-term ethanol self-administration induces ΔFosB in male and female adolescent, but not in adult, Wistar rats

Early-onset ethanol consumption predicts later development of alcohol use disorders. Age-related differences in reactivity to ethanol's effects may underlie this effect. Adolescent rats are more sensitive and less sensitive than adults to the appetitive and aversive behavioral effects of ethanol, respectively, and more sensitive to the neurotoxic effects of experimenter-administered binge doses of ethanol. However, less is known about age-related differences in the neural consequences of self-administered ethanol. ΔFosB is a transcription factor that accumulates after chronic drug exposure and serves as a molecular marker of neural plasticity associated with the transition to addiction. We analyzed the impact of chronic (18 two-bottle choice intake sessions spread across 42days, session length: 18h) ethanol [or only vehicle (control group)] self-administration during adolescence or adulthood on the induction of ΔFosB in several brain areas, anxiety-like behavior, and ethanol-induced locomotor activity and conditioned place preference (CPP) in Wistar rats. Adolescent rats exhibited a progressive escalation of ethanol intake and preference, whereas adult rats exhibited a stable pattern of ingestion. Few behavioral differences in the open field or light-dark test were observed after the intake test. Furthermore, ethanol self-administration did not promote the expression of ethanol-induced CPP. There were, however, large age-related differences in the neural consequences of ethanol drinking: a significantly greater number of ethanol-induced ΔFosB-positive cells was found in adolescents vs. adults in the prelimbic cortex, dorsolateral striatum, nucleus accumbens core and shell, and central amygdala nucleus capsular and basolateral amygdala, with sex-related differences found at central amygdala. This greater ethanol-induced ΔFosB induction may represent yet another age-related difference in the sensitivity to ethanol that may put adolescents at higher risk for problematic ethanol use.

An ultrastructural analysis of the effects of ethanol self-administration on the hypothalamic paraventricular nucleus in rhesus macaques.

A bidirectional relationship between stress and ethanol exists whereby stressful events are comorbid with problematic ethanol use and prolonged ethanol exposure results in adaptations of the physiological stress response. Endocrine response to stress is initiated in the hypothalamic paraventricular nucleus (PVN) with the synthesis and release of corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP). Alterations in CRH and AVP following long-term ethanol exposure in rodents is well demonstrated, however little is known about the response to ethanol in primates or the mechanisms of adaptation. We hypothesized that long-term ethanol self-administration in nonhuman primates would lead to ultrastructural changes in the PVN underlying adaptation to chronic ethanol. Double-label immunogold electron microscopy (EM) was used to measure presynaptic gamma-aminobutyric acid (GABA) and glutamate density within synaptic terminals contacting CRH- and AVP-immunoreactive dendrites. Additionally, pituitary-adrenal hormones (ACTH, cortisol, DHEA-s and aldosterone) under two conditions (low and mild stress) were compared before and after self-administration. All hormones were elevated in response to the mild stressor independent of ethanol consumption. The presynaptic glutamate density in recurrent (i.e., intra-hypothalamic) CRH terminals was highly related to ethanol intake, and may be a permissive factor in increased drinking due to stress. Conversely, glutamate density within recurrent AVP terminals showed a trend-level increase following ethanol, but was not related to average daily consumption. Glutamate density in non-recurrent AVP terminals was related to aldosterone under the low stress condition while GABAergic density in this terminal population was related to water consumption. The results reveal distinct populations of presynaptic terminals whose glutamatergic or GABAergic density were uniquely related to water and ethanol consumption and circulating hormones.

Disposition of d‐penicillamine, a promising drug for preventing alcohol‐relapse. Influence of dose, chronic alcohol consumption and age: studies in rats

Pharmacokinetic studies concerning d-penicillamine (an acetaldehyde sequestering agent) are scarce and have not evaluated the influence of chronic ethanol consumption and age on its disposition. Since recent preclinical studies propose d-penicillamine as a promising treatment for alcohol relapse, the main aim of the present work was to evaluate the influence of these two factors on d-penicillamine disposition in order to guide future clinical studies on the anti-relapse efficacy of this drug in alcoholism. Additionally, the effect of the administered dose was also evaluated. To this end, three studies were carried out. Study 1 assessed the influence of dose on d-penicillamine disposition, whereas studies 2 and 3 evaluated, respectively, the influence of chronic alcohol consumption and age. Rapid intravenous administrations of 2, 10 and 30 mg/kg of d-penicillamine were performed using young or adult ethanol-naïve rats or adult ethanol-experienced (subjected to a long-term ethanol self-administration protocol) rats. Pharmacokinetic parameters were derived from the biexponential model. Statistical analysis of CL, normalized AUC0 (∞) , V1 and k10 revealed that disposition, in the range plasma concentrations assayed, is non-linear both in young ethanol-naïve and in adult ethanol-experienced rats. Notably, no significant changes in t1/2 were detected. Chronic ethanol consumption significantly reduced CL values by 35% without affecting t1/2 . d-Penicillamine disposition was equivalent in young and adult animals. In conclusion, although DP pharmacokinetics is non-linear, the lack of significant alterations of the t1/2 would potentially simplify the clinical use of this drug. Chronic consumption of ethanol also alters d-penicillamine disposition but, again, does not modify t1/2.

Ethanol self-administration modulation of NMDA receptor subunit and related synaptic protein mRNA expression in prefrontal cortical fields in cynomolgus monkeys

Background: Functional impairment of the orbital and medial prefrontal cortex underlies deficits in executive control that characterize addictive disorders, including alcohol addiction. Previous studies indicate that alcohol alters glutamate neurotransmission and one substrate of these effects may be through the reconfiguration of the subunits constituting ionotropic glutamate receptor (iGluR) complexes. Glutamatergic transmission is integral to cortico-cortical and cortico-subcortical communication, and alcohol-induced changes in the abundance of the receptor subunits and/or their splice variants may result in critical functional impairments of prefrontal cortex in the alcohol-addicted state. Methods and results: The effects of chronic ethanol self-administration on glutamate receptor ionotropic NMDA (GRIN), as well as GRIN1 splice variant mRNA expression was studied in the orbitofrontal cortex (OFC; Area 13), dorsolateral prefrontal cortex (DLPFC; Area 46) and anterior cingulate cortex (ACC; Area 24) of male cynomolgus monkeys. Chronic ethanol self-administration resulted in significant changes in the expression of NMDA subunit mRNA expression in the DLPFC and OFC, but not the ACC. In DLPFC, the overall expression of NMDA subunits was significantly decreased in ethanol treated monkeys. Slight but significant changes were observed for synaptic associated protein 102 kD (SAP102) and neuronal nitric oxide synthase (nNOS) mRNAs. In OFC, the NMDAR1 variant GRIN1-1 was reduced while GRIN1-2 was increased. Furthermore, no significant changes in GFAP protein levels were observed in either the DLPFC or OFC. Conclusion: Results from these studies provide the first demonstration of posttranscriptional regulation of iGluR subunits in the primate brain following long-term ethanol self-administration. Furthermore, changes in these transcripts do not appear to reflect changes in glial activation or loss. Further studies examining the expression and cellular localization of subunit proteins and receptor pharmacology would shed more light on the findings reported here.

Circadian Timing of Ethanol Exposure Exerts Enduring Effects on Subsequent Ad Libitum Consumption in C57 Mice

There is a daily rhythm in the voluntary intake of ethanol in mice, with greatest consumption in the early night and lowest intake during the day. The role of daily timing of ethanol exposure on the development and control of long-term ethanol self-administration has been neglected. The present study examines these issues using C57BL/6J mice. Mice were repeatedly exposed to 10% ethanol for 2 hours early in the night or day for several weeks. Subsequently, ethanol was available at the opposite time (Expt 1) or 24 hours daily (Expts 1 and 2). Lick sensors recorded the patterns of drinking activity in Experiment 2. Mice exposed to ethanol during the night drink more than mice exposed during the day. Prior history did not affect ethanol intake when the schedule was reversed. Under 24-hour exposure conditions, mice with a history of drinking during the night consumed significantly more than mice drinking during the day. The circadian patterns of drinking were not altered. These results demonstrate that the daily timing of ethanol exposure exerts enduring effects of self-administration of ethanol in mice. Understanding how circadian rhythms regulate ethanol consumption may be valuable for modifying subsequent intake.

Long-term ethanol self-administration by cynomolgus macaques alters the pharmacology and expression of GABAA receptors in basolateral amygdala.

We have recently demonstrated that chronic ethanol ingestion alters the functional and pharmacological properties of GABAA receptors measured in acutely isolated rat lateral/basolateral amygdala neurons, a limbic forebrain region involved with fear-learning and innate anxiety. To understand relevance of these results in the context of primates, we have examined the effects of long-term ethanol self-administration on basolateral amygdala GABAA receptor pharmacology and expression in cynomolgus macaques (Macaca fascicularis). The impact of this 18-month-long exposure on GABAA receptor function was assessed in acutely isolated neurons from basolateral amygdala with whole-cell patch-clamp electrophysiology. Neurons from control animals expressed maximal current densities that were not significantly different from the maximal current densities of neurons from ethanol-treated animals. However, the GABA concentration-response relationships from ethanol-exposed neurons were significantly right-shifted compared with control neurons. These adaptations were associated with significant alterations in some characteristics of macroscopic current desensitization. To understand the mechanism governing these adaptations, we quantified GABAA alpha subunit mRNAs in basolateral amygdala from the same animals. mRNA levels of the alpha2 and alpha3 subunits were significantly decreased, whereas decreases in alpha1 expression only approached statistical significance. There were no changes in alpha4 mRNA levels. These findings indicate that ethanol-induced alterations in GABAA function may be regulated in part by selective changes in the expression of particular alpha subunits. We conclude that adaptations of basolateral amygdala GABAA receptors after long-term ethanol self-administration by the cynomolgus macaque are similar, but not identical, to those described in rodents after a brief forced ethanol exposure.