Chronic ethanol exposure facilitates facial-evoked MLI-PC synaptic transmission via nitric oxide signaling pathway in vivo in mice

We previously demonstrated that chronic ethanol exposure and withdrawal experience significantly increased subsequent voluntary ethanol intake in C57BL/6J mice. This study was conducted to examine chronic ethanol conditions that optimize this enhanced ethanol-drinking behavior. The purpose of this study was to examine whether the pattern and/or number of chronic ethanol exposures influence subsequent ethanol intake. C57BL/6J mice were trained to drink ethanol (15% v/v) in a limited access situation (2 h/day) until stable intake was achieved. In experiment 1, mice received two cycles of chronic ethanol exposure delivered either in an intermittent [multiple withdrawal (MW)] or continuous [continuous exposure (CE)] pattern. One week of daily drinking sessions followed each exposure. In experiment 2, mice received either two or four cycles of chronic intermittent ethanol exposure (MW), each followed by a week of testing sessions. Three additional weeks of ethanol intake testing followed the last ethanol (or air) exposure. Experiment 1: Only the MW group evidenced a significant increase in ethanol intake compared to controls after the first chronic ethanol exposure. Both MW and CE groups consumed more ethanol than controls after the second ethanol-exposure period. Experiment 2: Ethanol intake in MW mice compared to controls significantly increased after two or four cycles of chronic ethanol exposure/withdrawal, and this heightened ethanol intake lasted longer in mice that received four cycles of chronic intermittent ethanol exposure. Increased voluntary ethanol intake after chronic ethanol exposure and withdrawal experience may be accelerated by intermittent (as opposed to continuous) ethanol exposure, and the effect may last longer with increased number of such experiences.

Chronic, but not acute, ethanol exposure impairs central hypercapnic ventilatory drive in bullfrog tadpoles

After administration, ethanol and its metabolites go through the kidneys and are excreted into urine. The kidney seems to be the only vital organ generally spared in chronic alcoholics. Therefore, we investigated the multiple effects of chronic ethanol exposure on renal function tests and on oxidative stress related parameters in the kidney. Chronic ethanol (1.6 g ethanol/ kg body weight/ day) exposure did not show any significant change in relative weight (g/ 100g body weight) of kidneys, serum calcium level or glutathione s-transferase activity. However, urea and creatinine concentration in serum, and TBARS level in kidney elevated significantly, while reduced glutathione content and activities of glutathione peroxidase, glutathione reductase and superoxide dismutase diminished significantly after 12 weeks of ethanol exposure. Catalase activity showed increased activity after 4 weeks of ethanol exposure and decreased activity after 12 weeks of ethanol exposure. Genesis of renal ultrastructural abnormalities after 12 weeks of ethanol exposure may be important for the development of functional disturbances. This study revealed that chronic ethanol exposure for longer duration is associated with deleterious effects in the kidney.

Background:Chronic ethanol exposure reduces dopamine transmission in the nucleus accumbens, which may contribute to the negative affective symptoms associated with ethanol withdrawal. Kappa opioid receptors have been implicated in withdrawal-induced excessive drinking and anxiety-like behaviors and are known to inhibit dopamine release in the nucleus accumbens. The effects of chronic ethanol exposure on kappa opioid receptor-mediated changes in dopamine transmission at the level of the dopamine terminal and withdrawal-related behaviors were examined.Methods:Five weeks of chronic intermittent ethanol exposure in male C57BL/6 mice were used to examine the role of kappa opioid receptors in chronic ethanol-induced increases in ethanol intake and marble burying, a measure of anxiety/compulsive-like behavior. Drinking and marble burying were evaluated before and after chronic intermittent ethanol exposure, with and without kappa opioid receptor blockade by nor-binaltorphimine (10mg/kg i.p.). Functional alterations in kappa opioid receptors were assessed using fast scan cyclic voltammetry in brain slices containing the nucleus accumbens.Results:Chronic intermittent ethanol-exposed mice showed increased ethanol drinking and marble burying compared with controls, which was attenuated with kappa opioid receptor blockade. Chronic intermittent ethanol-induced increases in behavior were replicated with kappa opioid receptor activation in naïve mice. Fast scan cyclic voltammetry revealed that chronic intermittent ethanol reduced accumbal dopamine release and increased uptake rates, promoting a hypodopaminergic state of this region. Kappa opioid receptor activation with U50,488H concentration-dependently decreased dopamine release in both groups; however, this effect was greater in chronic intermittent ethanol-treated mice, indicating kappa opioid receptor supersensitivity in this group.Conclusions:These data suggest that the chronic intermittent ethanol-induced increase in ethanol intake and anxiety/compulsive-like behaviors may be driven by greater kappa opioid receptor sensitivity and a hypodopaminergic state of the nucleus accumbens.

The medial prefrontal cortex (mPFC) inhibits impulsive and compulsive behaviors that characterize drug abuse and dependence. Acamprosate is the leading medication approved for the maintenance of abstinence, shown to reduce craving and relapse in animal models and human alcoholics. Whether acamprosate can modulate executive functions that are impaired by chronic ethanol (EtOH) exposure is unknown. Here we explored the effects of acamprosate on an attentional set-shifting task and tested whether these behavioral effects are correlated with modulation of glutamatergic synaptic transmission and intrinsic excitability of mPFC neurons. We induced alcohol dependence in mice via chronic intermittent EtOH (CIE) exposure in vapor chambers and measured changes in alcohol consumption in a limited access 2-bottle choice paradigm. Impairments of executive function were assessed in an attentional set-shifting task. Acamprosate was applied subchronically for 2 days during withdrawal before the final behavioral test. Alcohol-induced changes in cellular function of layer 5/6 pyramidal neurons, and the potential modulation of these changes by acamprosate, were measured using patch clamp recordings in brain slices. Chronic EtOH exposure impaired cognitive flexibility in the attentional set-shifting task. Acamprosate improved overall performance and reduced perseveration. Recordings of mPFC neurons showed that chronic EtOH exposure increased use-dependent presynaptic transmitter release and enhanced postsynaptic N-methyl-D-aspartate receptor function. Moreover, CIE treatment lowered input resistance, and decreased the threshold and the after hyperpolarization of action potentials, suggesting chronic EtOH exposure also impacted membrane excitability of mPFC neurons. However, acamprosate treatment did not reverse these EtOH-induced changes cellular function. Acamprosate improved attentional control of EtOH exposed animals, but did not alter the concurrent changes in synaptic transmission or membrane excitability of mPFC neurons, indicating that these changes are not the pharmacological targets of acamprosate in the recovery of mPFC functions affected by chronic EtOH exposure.

A significant consequence of chronic ethanol (EtOH) exposure is the development of tolerance. The present study was designed to investigate tolerance to the discriminative stimulus properties of EtOH following chronic EtOH exposure. Adult male C57BL/6J mice were trained to discriminate EtOH (1.00 g/kg; i.p.) from saline, using a food-reinforced two-lever operant task. Following acquisition and establishment of criterion discrimination performance, a series of generalization tests were conducted to generate a baseline EtOH dose-response curve with a calculated ED50 dose of 0.42 g/kg. Mice were then placed into control (air) or EtOH inhalation chambers for 64 h. In Experiment 1, discriminative stimulus generalization tests with the EtOH ED50 dose conducted 24 h following chronic EtOH (or air) exposure did not yield significantly different EtOH responding, although a trend towards reduced sensitivity to the EtOH cue (tolerance) was evident. In Experiment 2, a cumulative dosing procedure (ED50=0.37 g/kg) was employed, yielding a baseline EtOH dose-response function with a calculated ED50 dose of 0.37 g/kg. At 24 h following chronic EtOH exposure, re-determination of the EtOH dose-response curve revealed a significant shift to the right, with more than a twofold increase in the ED50 value (ED50=1.09 g/kg) compared to the control air exposure condition (ED50=0.49 g/kg). This apparent tolerance to the EtOH cue dissipated in chronic EtOH-exposed mice tested 48 h following the inhalation treatment (ED50=0.51 g/kg). These results demonstrate tolerance to the discriminative stimulus effects of EtOH in C57BL/6J mice following chronic EtOH exposure in inhalation chambers.

Chronic ethanol exposure has been described in humans to produce a series of long and short term electrophysiological consequences. Interpretation of the electrophysiological findings in human subjects, however, is made difficult due to concomitant factors, such as nutritional status, premorbid functioning and differences in genetic susceptibility to the effects of ethanol. In the present study, electroencephalograms (EEGs) and auditory event related potentials (ERPs) were utilized to explore the short and longer term effects of chronic ethanol exposure in rats. Rats were continuously exposed to ethanol vapors for a period for 1 month. This treatment produced a mean blood ethanol level of 178 +/- 13.86 mg%. EEGs and ERPs were subsequently collected at 10 min, 24 h, and 2 weeks following termination of ethanol exposure. Significant changes in the EEGs and ERPs of these rats could be demonstrated. EEG amplitude increases, as quantified by spectral analysis, were most prominent at the 24 h time period, perhaps reflecting a state of "rebound excitability". EEG responses were normalized in ethanol-treated rats by 2 weeks post-withdrawal. In contrast, reductions in the N1 and P2 amplitudes of the rat ERPs were prominent after chronic ethanol exposure and following 2 weeks withdrawal, suggesting that ethanol may produce some longer term effects on response to ERP stimuli. Taken together, these studies suggest that ethanol may produce differential effects on EEG and ERPs and that this model may provide a useful substrate for the evaluation of the mechanisms underlying the effects of chronic ethanol exposure.

Given the prevalence of alcohol use in adolescence, it is important to understand the consequences of chronic ethanol exposure during this critical period in development. The purpose of this study was to assess possible age-related differences in susceptibility to tolerance development to ethanol-induced sedation and withdrawal-related anxiety, as well as voluntary ethanol intake after chronic exposure to relatively high doses of ethanol during adolescence or adulthood. Juvenile/adolescent and adult male Sprague-Dawley rats were assigned to one of five 10-day exposure conditions: chronic ethanol (4 g/kg every 48 hours), chronic saline (equivalent volume every 24 hours), chronic saline/acutely challenged with ethanol (4 g/kg on day 10), nonmanipulated/acutely challenged with ethanol (4 g/kg on day 10), or nonmanipulated. For assessment of tolerance development, duration of the loss of righting reflex (LORR) and blood ethanol concentrations (BECs) upon regaining of righting reflex (RORR) were tested on the first and last ethanol exposure days in the chronic ethanol group, with both saline and nonmanipulated animals likewise challenged on the last exposure day. Withdrawal-induced anxiety was indexed in a social interaction test 24 hours after the last ethanol exposure, with ethanol-naïve chronic saline and nonmanipulated animals serving as controls. Voluntary intake was assessed 48 hours after the chronic exposure period in chronic ethanol, chronic saline and nonmanipulated animals using an 8-day 2 bottle choice, limited-access ethanol intake procedure. In general, adolescent animals showed shorter durations of LORR and higher BECs upon RORR than adults on the first and last ethanol exposure days, regardless of chronic exposure condition. Adults, but not adolescents, developed chronic tolerance to the sedative effects of ethanol, tolerance that appeared to be metabolic in nature. Social deficits were observed after chronic ethanol in both adolescents and adults. Adolescents drank significantly more ethanol than adults on a gram per kilogram basis, with intake uninfluenced by prior ethanol exposure at both ages. Adolescents and adults may differ in their ability and/or propensity to adapt to chronic ethanol exposure, with adults, but not adolescents, developing chronic metabolic tolerance. However, this chronic exposure regimen was sufficient to disrupt baseline levels of social behavior at both ages. Taken together, these results suggest that, despite the age-related differences in tolerance development, adolescents are as susceptible as adults to consequences of chronic ethanol exposure, particularly in terms of disruptions in social behavior. Whether these effects would last into adulthood remains to be determined.

Alcohol use disorder (AUD) is characterized by compulsive drinking, which is thought to be mediated by effects of chronic intermittent ethanol exposure on the dorsal striatum, the input nucleus of the basal ganglia. Despite significant efforts to understand the impact of ethanol on the dorsal striatum, the rich diversity of striatal cell types and multitude of ethanol targets expressed by them necessitates an unbiased, discovery-based approach. In this study, we used single-nuclei RNA-sequencing (snRNA-seq; n = 86,715 cells) to examine the impact of chronic intermittent ethanol exposure on the dorsal striatum in C57BL/6 male and female mice. We detected 462 differentially expressed genes at FDR < 0.05, the majority of which were mapped to spiny projection neurons (SPNs), the most prominent cell type in the striatum. Gene co-expression network analysis and functional annotation of differentially expressed genes revealed down-regulation of postsynaptic intracellular signaling cascades in SPNs. Inflammation-related genes were down-regulated across many neuronal and non-neuronal cell types. Gene set enrichment analyses also pointed to altered states of rare cell types, including the induction of angiogenesis-related genes in vascular cells. A gene module down-regulated specifically in canonical SPNs was enriched for calcium-signaling genes and components of glutamatergic synapses, as well as for genes associated with genetic risk for AUD. Genetic perturbations of six of this module’s hub genes – Foxp1, Bcl11b, Pde10a, Rarb, Rgs9, and Itgr1 – had causal effects on its expression in the mouse striatum and/or on the broader set of differentially expressed genes in alcohol-exposed mice. These data provide important clues as to the impact of ethanol on striatal biology and provide a key resource for future investigation.

The central nucleus of the amygdala (CeA) is implicated in alcohol use disorder (AUD) and AUD-associated plasticity. The CeA is a primarily GABAergic nucleus that is subdivided into lateral and medial compartments with genetically diverse subpopulations. GABAA receptors are heteromeric pentamers with subunits conferring distinct physiological characteristics. GABAA receptor signaling in the CeA has been implicated in ethanol-associated plasticity; however, population-specific changes in inhibitory signaling and subunit expression remain unclear. Here, we combined electrophysiology with single-cell gene expression analysis of population markers and GABAA receptor subunits to examine population-specific changes in inhibitory control in male and female rats following chronic ethanol exposure. We found that chronic ethanol exposure and withdrawal produced global changes in GABAA receptor subunit expression at the transcript and protein levels, increased excitability in female CeA neurons, and increased inhibitory synaptic transmission in male CeA neurons. When we examined CeA neurons at the single-cell level we found heterogenous populations, as previously reported. We observed ethanol-induced increases in excitability only in somatostatin neurons in the CeA of females, decreases in excitability only in the protein kinase C delta (PKCd) population in males, and ethanol-induced increases in inhibitory transmission in male PKCd and calbindin 2-expressing CeA neurons. There were no population-specific differences in GABAA receptor (Gabr) subunits in males but reduced GabrA5 expression in female somatostatin neurons. Collectively, these findings suggest that defined CeA populations display differential ethanol sensitivity in males and females, which may play a role in sex differences in vulnerability to AUD or expression of AUD pathology.SIGNIFICANCE STATEMENT The CeA is involved in the effects of ethanol in the brain; however, the population-specific changes in CeA activity remain unclear. We used recordings of CeA neuronal activity and single-cell gene expression to examine population-specific changes in inhibitory control in male and female rats following chronic ethanol exposure and found sex- and population-specific effects of chronic ethanol exposure and withdrawal. Specifically, female CeA neurons displayed increased excitability in the somatostatin CeA population, whereas male CeA neurons displayed increased inhibitory control in both PKCd and calbindin populations and decreased excitability in the PKCd population. These findings identify CeA populations that display differential sensitivity to ethanol exposure, which may contribute to sex differences in vulnerability to alcohol use disorder.

Chronic plus binge ethanol exposure causes more severe pancreatic injury and inflammation

Effects of chronic ethanol consumption on rat GABA A and strychnine-sensitive glycine receptors expressed by lateral/basolateral amygdala neurons

Zinc Supplementation Prevents Alcoholic Liver Injury in Mice through Attenuation of Oxidative Stress

There is increasing evidence that heavy ethanol exposure in early life may produce long-lasting neurobehavioral consequences, since brain structural maturation continues until adolescence. It is well established that females are more susceptible to alcohol-induced neurotoxicity and that ethanol consumption is increasing among women, especially during adolescence. In the present study, we investigated whether chronic ethanol exposure during adolescence through early adulthood in female rats may induce hippocampal histological damage and neurobehavioral impairments. Female rats were treated with distilled water or ethanol (6.5 g/kg/day, 22.5% w/v) by gavage from the 35(th)-90(th) day of life. Ethanol-exposed animals displayed reduced exploration of the central area and increased number of fecal boluses in the open field test indicative of anxiogenic responses. Moreover, chronic high ethanol exposure during adolescence induced marked impairments on short-term memory of female rats addressed on social recognition and step-down inhibitory avoidance tasks. These neurobehavioral deficits induced by ethanol exposure during adolescence through early adulthood were accompanied by the reduction of hippocampal formation volume as well as the loss of neurons, astrocytes and microglia cells in the hippocampus. These results indicate that chronic high ethanol exposure during adolescence through early adulthood in female rats induces long-lasting emotional and memory deficits associated with morphological and molecular alterations in the hippocampus.

Ethanol exposure impairs mammalian reproductive function. However, the mechanisms are not fully understood. Adult female rats were given an ethanol or a calorically matched control diet. A third group was given a liquid nonethanol diet. Half the animals were killed at 2 weeks (short chronic) and the other half at 2 months (long chronic), all on the day of proestrous on the basis of daily vaginal smears. The major effect of ethanol feeding was disruption in the estrous cycle. Although all of the pair-fed animals continued to cycle, 40% of the ethanol rats in the short chronic study had disruption of their cycles. In the long chronic study, 83% of the ethanol animals had abnormal cycling, in contrast to 16% of the pair-fed controls. The nature of the cycle disruption was prolongation of diestrous, with an increased time interval between proestrous surges. In four ethanol-fed rats, there was complete cessation of the estrous cycle. However, ethanol did not decrease ovarian or uterine weight. Ethanol significantly increased serum estradiol in the short chronic but not long chronic study, whereas progesterone was unchanged. Ethanol did cause a significant reduction in circulating insulin-like growth factor. The major effect of both short chronic and long chronic ethanol exposure was disruption of the estrous regularity, leading to a decreased number of proestrous surges. Part of the mechanism of this disruption might be a transient estrogen increase or a decrease in circulating insulin-like growth factor.