Circulating and hepatic endocannabinoids and endocannabinoid‐related molecules in patients with cirrhosis

Background/Aims: Endocannabinoids include anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG). Endocannabinoid‐related molecules like oleoyl‐ethanolamine (OEA) and palmitoyl‐ethanolamine (PEA) have also been identified. AEA contributes to the pathogenesis of cardiovascular alterations in experimental cirrhosis, but data on the endocannabinoid system in human cirrhosis are lacking. Thus, we aimed to assess whether circulating and hepatic endocannabinoids are upregulated in cirrhotic patients and whether their levels correlate with systemic haemodynamics and liver function. Methods: The endocannabinoid levels were measured in peripheral and hepatic veins and liver tissue by isotope‐dilution liquid chromatography‐atmospheric pressure chemical ionization‐mass spectrometry. Systemic haemodynamics were assessed by the transthoracic electrical bioimpedance technique. Portal pressure was evaluated by hepatic venous pressure gradient. Results: Circulating AEA and, to a greater extent, PEA and OEA were significantly higher in cirrhotic patients than in controls. PEA and OEA were also increased in the cirrhotic liver tissue. AEA, OEA and PEA levels were significantly higher in peripheral than in the hepatic veins of cirrhotic patients, while the opposite occurred for 2‐AG. Finally, circulating AEA, OEA and PEA correlated with parameters of liver function, such as serum bilirubin and international normalized ratio. No correlations were found with systemic haemodynamics. Conclusions: The endocannabinoid system is upregulated in human cirrhosis. Peripheral AEA is increased in patients with a high model of end‐stage liver disease score and may reflect the extent of liver dysfunction. In contrast, the 2‐AG levels, the other major endocannabinoid, are not affected by cirrhosis. The upregulation of the endocannabinoid‐related molecules, OEA and PEA, is even greater than that of AEA, prompting pharmacological studies on these compounds.     

Reverse microdialysis of a dopamine uptake inhibitor in the nucleus accumbens of alcohol-preferring rats: effects on dialysate dopamine levels and ethanol intake

Background: The mesolimbic dopamine (DA) system has been implicated in mediating the reinforcing actions of ethanol (EtOH). This study examines the effects of local perfusion of the DA uptake inhibitor GBR12909 (GBR) on (1) DA levels in the nucleus accumbens (NAc) and (2) EtOH drinking in alcohol‐preferring rats. Methods: Stable drinking of a 15% (v/v) EtOH solution (minimum of 0.75 g/kg body weight) was established in daily 1 hr limited access sessions. Rats were then implanted with bilateral guide cannulae aimed 4 mm above the NAc. After recovery from surgery, concentric microdialysis probes (2 mm dialysis membrane surface) were inserted into the NAc. Most placements were in the shell or overlapping both shell and core. Two days later, the probes were perfused at 1.0 μl/min with artificial cerebral spinal fluid (aCSF) for at least a 90 min washout period followed by collection of five basal samples over 150 min. Rats were then perfused with either aCSF alone or 10, 25, 100, or 200 μM of GBR for 240 min on the first day of microdialysis. During the last 60 min of the drug treatment phase, rats were given their scheduled access to 15% EtOH. All rats were then perfused with aCSF for the last 90 min of the experiment. The following day, the procedure was repeated, but animals that received aCSF on the first day were given a dose of GBR and rats given GBR on the first day received only aCSF. Results: GBR perfusion increased extracellular NAc DA levels dose dependently to more than 800% of basal levels at 100 to 200 μM but failed to alter EtOH intake (p > 0.05, paired t test) at any concentration tested. Moreover, after 100 μM of GBR perfusion had terminated, the extracellular levels of DA in the NAc remained elevated for approximately 24 hr (790% of day 1 basal;p < 0.05). The increase in dialysate DA levels observed during GBR perfusion with 100 μM was significantly greater for EtOH‐experienced rats than for EtOH‐naïve rats [F (7,59) = 14.85, p < 0.0001, analysis of variance, Student‐Newman‐Keuls post hoc test]. Conclusions: The results suggest (1) that EtOH drinking experience induces neuroadaptations that increase DA release in the NAc, and (2) that additional elevation in synaptic levels of DA in the NAc does not influence the maintenance of ongoing alcohol drinking under scheduled access conditions in alcohol‐preferring animals.     

Altered circulating endocannabinoids in anorexia nervosa during acute and weight‐restored phases: A pilot study

Anorexia nervosa (AN) is an eating disorder characterized by a low food intake and often exceeding exercise, leading to a particularly low body × weight proportion. Patients with AN usually report less hunger than healthy controls. Endogenous endocannabinoids (eCBs), specifically the anandamide, have been associated to hunger, as a meal initiator, but research regarding AN and eCB and inconclusive. In this pilot study, we investigated plasma levels of eCB in inpatients with AN during fasting and after eating, both during the acute AN phase and after weight recovery. After an 8‐hr fasting period, blood sample was collected from all participants. After that, participants were given a muffin test meal. Blood samples for the investigation of endogenous eCBs anandamide (N‐arachidonoylethanolamide [AEA]) and 2‐arachidonoylglycerol (2‐AG) were then collected after 120 and 240 min. Participants were only allowed to eat and drink what was offered them during the research. AN reported less hunger than controls during fasting and at the end of the experiment. Also, plasma levels of AEA were significantly smaller in AN in comparison with controls in all time points. No significant difference was found for 2‐AG plasma levels. After recovery, no significant difference was found for eCB levels. These findings could be interpreted as an AEA deregulation in AN before and after food intake, which persists after weight recovery. These findings may have implications to the pharmacological treatment of AN and to relapse occurring in the disorder.     

Suppression of Heavy Drinking and Alcohol Seeking by a Selective ALDH-2 Inhibitor

Background: Inherited human aldehyde dehydrogenase 2 (ALDH‐2) deficiency reduces the risk for alcoholism. Kudzu plants and extracts have been used for 1,000 years in traditional Chinese medicine to treat alcoholism. Kudzu contains daidzin, which inhibits ALDH‐2 and suppresses heavy drinking in rodents. Decreased drinking due to ALDH‐2 inhibition is attributed to aversive properties of acetaldehyde accumulated during alcohol consumption. However, daidzin can reduce drinking in some rodents without necessarily increasing acetaldehyde. Therefore, a selective ALDH‐2 inhibitor might affect other metabolic factors involved in regulating drinking. Methods: Aldehyde dehydrogenase 2 inhibitors were synthesized based on the co‐crystal structure of ALDH‐2 and daidzin. We tested the efficacy of a highly selective reversible ALDH‐2 inhibitor, CVT‐10216, in models of moderate and high alcohol drinking rats. We studied 2‐bottle choice and deprivation‐induced drinking paradigms in Fawn Hooded (FH) rats, operant self‐administration in Long Evans (LE), FH, and inbred P (iP) rats and in cue‐induced reinstatement in iP rats. We also assayed blood acetaldehyde levels as well as dopamine (DA) release in the nucleus accumbens (NAc) and tested possible rewarding/aversive effects of the inhibitor in a conditioned place preference (CPP) paradigm. Results: CVT‐10216 increases acetaldehyde after alcohol gavage and inhibits 2‐bottle choice alcohol intake in heavy drinking rodents, including deprivation‐induced drinking. Moreover, CVT‐10216 also prevents operant self‐administration and eliminates cue‐induced reinstatement of alcohol seeking even when alcohol is not available (i.e., no acetaldehyde). Alcohol stimulates DA release in the NAc, which is thought to contribute to increased drinking and relapse in alcoholism. CVT‐10216 prevents alcohol‐induced increases in NAc DA without changing basal levels. CVT‐10216 does not show rewarding or aversive properties in the CPP paradigm at therapeutic doses. Conclusion: Our findings suggest that selective reversible ALDH‐2 inhibitors may have therapeutic potential to reduce excessive drinking and to suppress relapse in abstinent alcoholics.     

Effects of ethanol exposure on subsequent acquisition and extinction of ethanol self-administration and expression of alcohol-seeking behavior in adult alcohol-preferring (P) rats: I. Periadolescent exposure

Background The current study examined the effects of ethanol (EtOH) drinking during periadolescence on the subsequent acquisition and extinction of operant self‐administration of EtOH and expression of alcohol‐seeking behavior in adult alcohol‐preferring (P) rats to test the hypothesis that alcohol drinking during periadolescence produces enduring alterations that enhance the reinforcing properties of EtOH. Methods Periadolescent female P rats were given 24 hr free‐choice access to 15% (v/v) EtOH starting at postnatal day (PND) 30 and ending on PND 60 or were similarly housed and received water only. On PND 75, without any prior training, periadolescent alcohol‐drinking and periadolescent alcohol‐naïve rats were placed in standard two‐lever (15% EtOH and water) chambers to examine acquisition of EtOH self‐administration with a fixed ratio (FR) 1 schedule of reinforcement. After the acquisition phase and after stable responding was established on an FR5 for EtOH and FR1 for water, P rats underwent extinction training for both EtOH and water rewards. After extinction training and a 2 week home cage period, rats were returned to the operant chambers in the absence of reward for seven consecutive sessions (Pavlovian spontaneous recovery). After this testing period, animals were maintained in their home cage for a week before being returned to the operant chambers and allowed to respond for EtOH and water (reacquisition). Results Compared with periadolescent alcohol‐naïve rats, periadolescent alcohol‐drinking rats acquired EtOH responding sooner (i.e., in the first acquisition session), displayed a greater resistance to extinguish EtOH responding (i.e., higher levels of responding in sessions 4–6), had higher responding for more sessions on the EtOH lever in the absence of reward after a prolonged home cage rest period, and had a more prolonged elevated level of EtOH responding during reacquisition (four sessions versus one session). Conclusions Overall, the results suggest that periadolescent EtOH drinking by P rats produced long‐lasting alterations in the reinforcing effects of alcohol, which increased the likelihood that alcohol drinking would be initiated in adulthood, decreased the likelihood that once adult alcohol drinking began it could be extinguished easily, and increased the potential for relapse.     

Limited Access Alcohol Drinking in High- and Low-Alcohol Preferring Selected Lines of Mice

BACKGROUND: Selection studies and genetic analyses of drinking behavior in rodents often involved unlimited access to alcohol over a period of weeks, with water and food freely available. Most studies investigating the pharmacology of alcohol drinking, on the other hand, use procedures in which access to alcohol is limited to a particular time each day. Reconciliation of findings between these two conditions likely depends on their sharing common genetic mechanisms as indicated, for example, by covariation in response to selection. To this end, high- and low-alcohol preferring (HAP and LAP, respectively) mice, selected for differences in 24-hr access alcohol drinking over a 4-week period, were subjected to a limited access alcohol drinking protocol. METHODS: During 2-hr sessions, mice had access to various concentrations of alcohol (7-15%, v/v) in the home cage for 2 hr a day, with ad libitum access to food and water. Additional sessions were conducted with no food present. RESULTS: Although both strains consumed alcohol and water during these sessions, HAP mice drank far more alcohol than did LAP mice. HAP but not LAP mice drank alcohol at a high rate early in the session compared with later in the session. Additionally, HAP mice responded to changes in alcohol concentration, whereas LAP mice did not. Removal of food did not influence alcohol drinking, although water drinking decreased following food removal. HAP mice reached appreciable blood alcohol concentrations after limited access. CONCLUSIONS: These findings indicate that in these selectively bred mice, alcohol drinking during limited and unlimited access may be genetically related, and that drinking during limited access sessions in HAP mice is likely for the pharmacological properties of alcohol.     

Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats

Background: There has been some difficulty getting standard laboratory rats to voluntarily consume large amounts of ethanol without the use of initiation procedures. It has previously been shown that standard laboratory rats will voluntarily consume high levels of ethanol if given intermittent‐access to 20% ethanol in a 2‐bottle‐choice setting [ Wise, Psychopharmacologia 29 (1973), 203 ]. In this study, we have further characterized this drinking model. Methods: Ethanol‐naïve Long–Evans rats were given intermittent‐access to 20% ethanol (three 24‐hour sessions per week). No sucrose fading was needed and water was always available ad libitum. Ethanol consumption, preference, and long‐term drinking behaviors were investigated. Furthermore, to pharmacologically validate the intermittent‐access 20% ethanol drinking paradigm, the efficacy of acamprosate and naltrexone in decreasing ethanol consumption were compared with those of groups given continuous‐access to 10 or 20% ethanol, respectively. Additionally, ethanol consumption was investigated in Wistar and out‐bred alcohol preferring (P) rats following intermittent‐access to 20% ethanol. Results: The intermittent‐access 20% ethanol 2‐bottle‐choice drinking paradigm led standard laboratory rats to escalate their ethanol intake over the first 5 to 6 drinking sessions, reaching stable baseline consumption of high amounts of ethanol (Long–Evans: 5.1 ± 0.6; Wistar: 5.8 ± 0.8 g/kg/24 h, respectively). Furthermore, the cycles of excessive drinking and abstinence led to an increase in ethanol preference and increased efficacy of both acamprosate and naltrexone in Long–Evans rats. P‐rats initiate drinking at a higher level than both Long–Evans and Wistar rats using the intermittent‐access 20% ethanol paradigm and showed a trend toward a further escalation in ethanol intake over time (mean ethanol intake: 6.3 ± 0.8 g/kg/24 h). Conclusion: Standard laboratory rats will voluntarily consume ethanol using the intermittent‐access 20% ethanol drinking paradigm without the use of any initiation procedures. This model promises to be a valuable tool in the alcohol research field.     

Neuroadaptations of Cdk5 in cholinergic interneurons of the nucleus accumbens and prefrontal cortex of inbred alcohol-preferring rats following voluntary alcohol drinking

BACKGROUND: Neurobiological studies have identified brain areas and related molecular mechanisms involved in alcohol abuse and dependence. Specific cell types in these brain areas and their role in alcohol-related behaviors, however, have not yet been identified. This study examined the involvement of cholinergic cells in inbred alcohol-preferring rats following 1 month of alcohol drinking. Cyclin-dependent kinase 5 (Cdk5) immunoreactivity (IR), a marker of neuronal plasticity, was examined in cholinergic neurons of the nucleus accumbens (NuAcc) and prefrontal cortex (PFC) and other brain areas implicated in alcohol drinking, using dual immunocytochemical (ICC) procedures. Single Cdk5 IR was also examined in several brain areas implicated in alcohol drinking. METHODS: The experimental group self-administered alcohol using a 2-bottle-choice test paradigm with unlimited access to 10% (v/v) alcohol and water for 23 h/d for 1 month. An average of 6 g/kg alcohol was consumed daily. Control animals received identical treatment, except that both bottles contained water. Rats were perfused and brain sections were processed for ICC procedures. RESULTS: Alcohol drinking resulted in a 51% increase in Cdk5 IR cholinergic interneurons in the shell NuAcc, while in the PFC there was a 51% decrease in the percent of Cdk5 IR cholinergic interneurons in the infralimbic region and a 46% decrease in Cdk5 IR cholinergic interneurons in the prelimbic region. Additionally, single Cdk5 IR revealed a 42% increase in the central nucleus of the amygdala (CNA). CONCLUSIONS: This study identified Cdk5 neuroadaptation in cholinergic interneurons of the NuAcc and PFC and in other neurons of the CNA following 1 month of alcohol drinking. These findings contribute to our understanding of the cellular and molecular basis of alcohol drinking and toward the development of improved region and cell-specific pharmacotherapeutic and behavioral treatment programs for alcohol abuse and alcoholism.     

Corticotropin-releasing factor acting on corticotropin-releasing factor receptor type 1 is critical for binge alcohol drinking in mice

Background: The corticotropin‐releasing factor (CRF) system has been implicated in the regulation of alcohol consumption. However, previous mouse knockout (KO) studies using continuous ethanol access have failed to conclusively confirm this. Recent studies have shown that CRF receptor type 1 (CRFR1) antagonists attenuate alcohol intake in the limited access “drinking in the dark” (DID) model of binge drinking. To avoid the potential nonspecific effects of antagonists, in this study, we tested alcohol drinking in CRFR1, CRFR2, CRF, and urocortin 1 (Ucn1) KO and corresponding wild‐type (WT) littermates using the DID paradigm. Methods: On days 1 to 3, the CRFR1, CRFR2, Ucn1, and CRF KO mice and their respective WT littermates were provided with 20% ethanol or 10% sucrose for 2 hours with water available at all other times. On day 4, access to ethanol or sucrose was increased to 4 hours. At the end of each drinking session, the volume of ethanol consumed was recorded, and at the conclusion of the last session, blood was also collected for blood ethanol concentration (BEC) analysis. Results: CRFR1 KO mice had lower alcohol intakes and BECs and higher intakes of sucrose compared with WTs. In contrast, CRFR2 KO mice, while having reduced intakes initially, had similar alcohol intakes on days 2 to 4 and similar BECs as the WTs. To determine the ligand responsible, Ucn1 and CRF KO and WT mice were tested next. While Ucn1 KOs had similar alcohol intakes and BECs to their WTs, CRF KO mice showed reduced alcohol consumption and lower BECs compared with WTs. Conclusions: Our results confirm that CRFR1 plays a key role in binge drinking and identify CRF as the ligand critically involved in excessive alcohol consumption.     

DRD2 gene transfer into the nucleus accumbens core of the alcohol preferring and nonpreferring rats attenuates alcohol drinking

BACKGROUND: Transient overexpression of the dopamine D2 receptor (DRD2) gene in the nucleus accumbens (NAc) using an adenoviral vector has been associated with a significant decrease in alcohol intake in Sprague Dawley rats. This overexpression of DRD2 reduced alcohol consumption in a two-bottle-choice paradigm and supported the view that high levels of DRD2 may be protective against alcohol abuse. METHODS: Using a limited access (1 hr) two-bottle-choice (water versus 10% ethanol) drinking paradigm, we examined the effects of the DRD2 vector in alcohol intake in the genetically inbred alcohol-preferring (P) and -nonpreferring (NP) rats. In addition, micro-positron emission tomography imaging was used at the completion of the study to assess in vivo the chronic (7 weeks) effects of ethanol exposure on DRD2 levels between the two groups. RESULTS: P rats that were treated with the DRD2 vector (in the NAc) significantly attenuated their alcohol preference (37% decrease) and intake (48% decrease), and these measures returned to pretreatment levels by day 20. A similar pattern of behavior (attenuation of ethanol drinking) was observed in NP rats. Analysis of the [C]raclopride micro-positron emission tomography data after chronic (7 weeks) exposure to ethanol revealed clear DRD2 binding differences between the P and NP rats. P rats showed 16% lower [C]raclopride specific binding in striatum than the NP rats. CONCLUSIONS: These findings further support our hypothesis that high levels of DRD2 are causally associated with a reduction in alcohol consumption and may serve as a protective factor against alcoholism. That this effect was seen in P rats, which are predisposed to alcohol intake, suggests that they are protective even in those who are genetically predisposed to high alcohol intake. It is noteworthy that increasing DRD2 significantly decreased alcohol intake but did not abolish it, suggesting that high DRD2 levels may specifically interfere with the administration of large quantities of alcohol. The significantly higher DRD2 concentration in NP than P rats after 7 weeks of ethanol therefore could account for low alcohol intake.     

Innate differences in the expression of brain-derived neurotrophic factor in the regions within the extended amygdala between alcohol preferring and nonpreferring rats

Background: Animal lines such as alcohol‐preferring (P) and nonpreferring (NP) rats appear to be suitable animal models to investigate the biological basis of alcohol‐drinking behaviors. The extended amygdala serves as a neuroanatomical substrate for alcohol‐drinking behaviors. Brain‐derived neurotrophic factor (BDNF) in the amygdala has been implicated in alcohol‐drinking behaviors; however, its expression in the extended amygdala of P and NP rats is unknown. Therefore, we examined the basal expression of BDNF in the extended amygdala of alcohol naïve P and NP rats. Methods: We determined the basal mRNA and protein levels of BDNF by in situ RT‐PCR and immuno‐histochemical procedure, respectively, in the amygdaloid [central nucleus of amygdala (CeA), medial nucleus of amygdala (MeA), and basolateral amygdala (BLA)], nucleus accumbal (NAc shell and core), and bed nucleus of stria terminalis (BNST) [lateral BNST (lBNST), medial BNST (mBNST), and ventral BNST (vBNST)] brain structures of P and NP rats. In addition, we examined the localization of BDNF in neurons using double‐immunofluorescence labeling of BDNF with neuron‐specific nuclear protein (NeuN) and also determined the number of NeuN‐positive neurons in the amygdaloid structures of P and NP rats. Results: The mRNA and protein levels of BDNF were found to be significantly lower in both the CeA and MeA, but not in the BLA, of P compared with NP rats. We also found that BDNF was expressed in neurons in the amygdaloid structures of P and NP rats. In addition, we found that the number of NeuN‐positive neurons was similar in the amygdaloid structures of P and NP rats. Interestingly, the mRNA and protein levels of BDNF were also significantly lower in the lBNST, mBNST, and vBNST of P compared with NP rats. On the other hand, mRNA and protein levels of BDNF were similar in the NAc shell and core structures of P and NP rats. Conclusions: P and NP rats are selectively bred for higher and lower alcohol preference, respectively; therefore it is possible that lower BDNF levels in the amygdaloid and BNST structures may be associated with the excessive alcohol‐drinking behaviors of P rats.     

Environmental cues, alcohol seeking, and consumption in baboons: effects of response requirement and duration of alcohol abstinence

BACKGROUND: Environmental stimuli (cues) that have been paired with alcohol drinking may evoke classically conditioned states that in turn influence alcohol consumption and relapse to heavy drinking. Animal models using chained schedules of alcohol reinforcement may be useful for examining such complex interactions. METHODS: Alcohol drinking was established in 4 baboons. A sequence of lights and tones was presented during daily 3-hour sessions. First, cues were presented alone and no programmed contingencies were in effect. Second, cues were paired with 3 linked components consisting of different behavioral contingencies leading to and concluding with access to alcohol for self-administration in the last component (i.e., a chained schedule of alcohol reinforcement). Third, the effects of withholding alcohol access (i.e., forced abstinence) and increasing the number of lever responses required per drink were evaluated. RESULTS: Cues paired with a chained schedule of alcohol reinforcement engendered behaviors that brought baboons into contact with alcohol-related cues and occasioned operant responding that facilitated access to alcohol (alcohol seeking) during components that preceded alcohol access. Increasing the response requirement for each drink decreased the number of drinks and volume of alcohol consumed, but did not alter alcohol seeking. On the first session after 14 days of alcohol abstinence, latency to complete the operant requirement that produced alcohol access was decreased while both alcohol self-administration and volume of alcohol consumed were increased. CONCLUSIONS: Alcohol self-administration and consumption were sensitive to increases in response requirement and duration of alcohol abstinence, while seeking was only enhanced by duration of alcohol abstinence. This animal model may be useful to further examine the interactions between environmental cues and behaviors associated with seeking and consumption of alcohol and to evaluate the efficacy of potential alcohol treatment drugs on these behaviors.     

Ethanol drinking experience attenuates (-)sulpiride-induced increases in extracellular dopamine levels in the nucleus accumbens of alcohol-preferring (P) rats

BACKGROUND: The reinforcing properties of ethanol may be partly mediated through the mesolimbic dopamine (DA) system. This study examines the effects of local application of the DA D(2) receptor antagonist (-)sulpiride (SUL) on ethanol drinking of alcohol-preferring (P) rats, and extracellular DA levels in the nucleus accumbens (NAc) of P rats that were either ethanol-naive or had been chronically drinking ethanol. METHODS: Microdialysis was used to sample NAc DA levels, and reverse microdialysis was used to locally administer the D(2) antagonist (-)sulpiride (SUL) into the NAc of adult female P rats that were either drinking ethanol (n = 17) or were ethanol-naive (n = 24). Stable intake of 15% (v/v) ethanol (>/=0.75 g/kg) was established for the ethanol-drinking group in daily 1-hr access periods over a minimum of 4 weeks before surgery. Naive and ethanol-drinking rats were implanted with bilateral guide cannulae aimed 4 mm above the NAc shell. After recovery from surgery, microdialysis probes (active area = 2 mm) were inserted bilaterally into the NAc. Two days later, rats in the ethanol-drinking and naive groups were each divided into two groups; one group was bilaterally perfused (1.0 microl/min) with artificial cerebrospinal fluid (aCSF) and the other group was further divided into three subgroups that were perfused with aCSF + either 50, 100, or 200 microM SUL for 240 min. During the last 60 min of perfusion, the ethanol-drinking rats were given their daily 1-hr ethanol access period. Following ethanol access, the aCSF + SUL subgroups were then given aCSF only. The entire perfusion procedure was repeated 24 hr later, but the aCSF only and aCSF + SUL group treatment conditions were transposed. RESULTS: In ethanol-drinking rats, 100 and 200 microM SUL increased extracellular NAc DA levels to approximately 200% of basal values, but did not significantly alter ethanol intake. In ethanol-naive P rats, 100 and 200 microM SUL increased extracellular NAc DA levels significantly more (450% of basal; p < 0.05) than in the ethanol-drinking group. CONCLUSIONS: The findings are consistent with the hypothesis that ethanol-drinking experience causes a desensitization or a down-regulation of D(2) autoreceptors in the NAc of P rats.     

Behavioral traits predicting alcohol drinking in outbred rats: an investigation of anxiety, novelty seeking, and cognitive flexibility

Background: Most adults in Western society consume alcohol regularly without negative consequences. For a small subpopulation, however, drinking can quickly progress to excessive and chronic intake. Given the dangers associated with alcohol abuse, it is critical to identify traits that may place an individual at risk for developing these behaviors. To that end, we used a rat model to determine whether anxiety‐related behaviors, novelty seeking, or cognitive flexibility predict excessive alcohol drinking under both limited and continuous access conditions. Methods: Adult male rats were assessed in a series of behavioral tasks (elevated plus maze [EPM], locomotor activity, and discrimination/reversal learning in a Y‐maze) followed by 6 weeks of daily, 1‐hour access to alcohol in a free‐choice, 2‐bottle paradigm (10% alcohol vs. tap water). Next, subjects were given the opportunity to consume alcohol for 72 hours in drinking chambers that permit separate measures of each drinking bout. Half of the animals experienced a 2‐week deprivation period between the limited and continuous access sessions. Results: Time spent on the open arms of the EPM, but not novelty seeking or discrimination/reversal learning, predicted alcohol consumption during limited, 1‐h/d access sessions to alcohol. Anxiety‐related behavior also predicted the escalation of intake when animals were given 72 hours of continuous access to alcohol. Bout size, but not frequency, was responsible for the increased consumption by high‐anxiety subjects during this period. Finally, intake during limited access sessions predicted intake during continuous access, but only in subjects with low intake during limited access. Conclusions: These findings confirm that preexisting anxiety‐related behavior predicts alcohol intake under several schedules of alcohol access. Moreover, when access is unlimited, the high‐anxiety‐related group exhibited an increase in bout size, but not frequency, of drinking. In addition, we show that modest intake when alcohol is restricted may or may not progress to excessive intake when the drug is freely available.     

Aripiprazole effects on alcohol consumption and subjective reports in a clinical laboratory paradigm--possible influence of self-control

Introduction: There has been increasing interest in the use of medications that affect the dopamine receptor in the treatment of alcoholism. Aripiprazole has the unique pharmacology of being a partial dopamine agonist serving to stabilize brain dopamine systems in both frontal cortical and subcortical areas. As such, it might act to dampen alcohol reinforcement and craving and/or alter control over alcohol use. The current clinical laboratory study was conducted to evaluate the safety and efficacy of aripiprazole as a potential agent to alter drinking and objective effects of alcohol. Methods: Thirty nontreatment seeking alcoholics were enrolled in a subacute human laboratory study and received double‐blind treatment with up to 15 mg of aripiprazole (n = 15) or identical placebo (n = 15) for 8 days. Tolerability and utility of aripiprazole was monitored during natural drinking over the first 6 days of medication treatment and also during a free choice limited access alcohol consumption paradigm following an initial drink of alcohol in a bar‐lab setting on Day 8. Results: Aripiprazole was well tolerated and reduced drinking in nontreatment seeking alcoholics over 6 days of natural drinking—especially in those with lower self control (more impulsive). It also reduced drinks in the bar‐lab after a priming drink and broke the link between priming drink induced stimulation and further drinking. During the bar‐lab drinking session, there were no differences in subjective high, intoxication, or craving between subjects treated with aripiprazole or placebo. Discussion: This study joins several others in demonstrating the utility of subacute dosing laboratory paradigms for evaluating medication effects in alcoholics. Aripiprazole was well tolerated and lowered alcohol use, especially in those with lower impulse control. Further study is needed to determine the safety and utility of aripiprazole in the treatment of alcoholism and if subgroups of alcoholics are more likely to respond.     

Consistent, high-level ethanol consumption in pig-tailed macaques via a multiple-session, limited-intake, oral self-dosing procedure

Background:  Alcohol abuse is a major public health burden that can lead to many adverse health effects such as impaired hepatic, gastrointestinal, central nervous system and immune system function. Preclinical animal models of alcohol abuse allow for experimental control over variables often difficult to control in human clinical studies (e.g., ethanol exposure before or during the study, history of other drug use, access to medical care, nutritional status, etc). Nonhuman primate models in particular provide increased genetic, anatomic and physiologic similarity to humans, relative to rodent models. A small percentage of macaques will spontaneously consume large quantities of ethanol; however, most nonhuman primate models of "voluntary" ethanol intake produce relatively low daily ethanol intake in the majority of monkeys.
Methods:  To facilitate study of chronic exposure to high levels of ethanol intake, a macaque model has been developed that induces consistent, daily high-level ethanol consumption. This multiple-session procedure employed 4 drinking sessions per day, with sessions occurring once every 6 hours.
Results:  The group average alcohol consumption was 4.6 g/kg/d (SEM 0.4), roughly twice the group average consumption of previous reports. Ethanol drinking sessions produced group mean blood ethanol levels of 95 mg/dl after 60 minutes, and fine motor control was impaired up to 90 minutes after a drinking session.
Conclusion:  This model of multiple-session, limited access, oral ethanol self-dosing produced consistent, high-level ethanol consumption with each session qualifying as a "binge" drinking session using the definition of "binge" provided by the NIAAA (>80 mg/dl/session). This model of ethanol drinking in macaques will be of great utility in the study of immunological, physiological and behavioral effects of ethanol in nonhuman primates.     

Effects of long-term episodic access to ethanol on the expression of an alcohol deprivation effect in low alcohol-consuming rats

Background: The alcohol‐preferring (P) and ‐nonpreferring (NP) and high alcohol–drinking (HAD) and low alcohol–drinking (LAD) rats have been selectively bred for divergent preference for ethanol over water. In addition, both P and HAD rats display an alcohol deprivation effect (ADE). This study was undertaken to test whether the NP, LAD‐1, and LAD‐2 lines of rats could display an ADE as well. Method: Adult female NP, LAD‐1, and LAD‐2 rats were given concurrent access to multiple concentrations of ethanol [5, 10, 15% (v/v)] and water in an ADE paradigm involving an initial 6 weeks of 24‐hr access to ethanol, followed by four cycles of 2 weeks of deprivation from and 2 weeks of re‐exposure to ethanol (5, 10, and 15%). A control group had continuous access to the ethanol concentrations (5, 10, and 15%) and water through the end of the fourth re‐exposure period. Results: For NP rats, a preference for the highest ethanol concentration (15%) was evident by the end of the fifth week of access (～60% of total ethanol fluid intake). Contrarily, LAD rats did not display a marked preference for any one concentration of ethanol. All three lines displayed an ADE after repeated cycles of re‐exposure to ethanol, with the general ranking of intake being LAD‐1 > NP > LAD‐2 (e.g., for the first day of reinstatement of the third re‐exposure cycle, intakes were 6.5, 2.9, and 2.4 g/kg/day compared with baseline values of 3.1, 2.0, and 1.3 g/kg/day for each line, respectively). By the 13th week, rats from all three lines, with a ranking of LAD‐1 > NP > LAD‐2, were drinking more ethanol (3.3, 2.2, and 2.0 g/kg/day, respectively) compared with their consumption during the first week of access (～1.1 g/kg/day for all three lines). Conclusion: These data indicate that access to multiple concentrations of ethanol and exposure to multiple deprivation cycles can partially overcome a genetic predisposition of NP, LAD‐1, and LAD‐2 rats for low alcohol consumption. In addition, the findings suggest that genetic control of low alcohol consumption in rats is not associated with the inability to display an ADE.     

Removal of 2-arachidonylglycerol by direct hemoperfusion therapy with polymyxin B immobilized fibers benefits patients with septic shock

Arachidonylethanolamide (AEA) and 2-arachidonylglycerol (2-AG) are endocannabinoids involved in septic shock, and 8-epi prostaglandin F2alpha (F2-isoprostane) is a biomarker of oxidative stress in biological systems. Because the antibiotic polymyxin B absorbs endocannabinoids as well as endotoxins, direct hemoperfusion therapy with polymyxin B-immobilized fibers (PMX-DHP) decreases serum levels of endocannabinoids. To investigate the features of sepsis and determine the proper use of PMX-DHP, we measured the changes in levels of endocannabinoids and F2-isoprostane in patients with septic shock. Twenty-six patients with septic shock, including those with septic shock induced by peritonitis, underwent laparotomy for drainage. Endocannabinoids absorption with PMX-DHP was examined in two groups of patients: patients whose mean arterial blood pressure (mABP) had increased more than 20 mm Hg (responder group; N = 13); and patients iwhose mABP did not increase or had increased no more than 20 mm Hg (non-responder group; N = 13). Levels of AEA did not change after PMX-DHP in either the non-responder or responder groups, whereas levels of 2-AG decreased significantly after PMX-DHP in the responder group, but not in the non-responder group. F2-isoprostane gradually increased after PMX-DHP treatment; on the other hand, levels of F2-isoprostane remained constant in the responder group. Patients with septic shock are under considerable oxidative stress, and 2-AG plays an important role in the cardiovascular status of these patients. The removal of 2-AG by PMX-DHP benefits patients with septic shock by stabilizing cardiovascular status and decreasing long-term oxidative stress.     

Involvement of arginine vasopressin and V1b receptor in alcohol drinking in Sardinian alcohol-preferring rats

Background: Recent animal studies have shown that the level of stress‐responsive arginine vasopressin (AVP) gene expression in the amygdala is increased during early withdrawal from long‐term heroin or cocaine administration. The selective AVP V1b receptor antagonist SSR149415 (capable of exerting antidepressant‐like and anxiolytic effects in animal models) also blocked stress‐induced reinstatement of drug‐seeking behavior. This study was undertaken to investigate the effects of alcohol and to determine whether (i) there are genetically determined differences in basal AVP mRNA levels in the medial/central amygdala (Me/CeA) and medial hypothalamus (MH) between selectively bred Sardinian alcohol‐preferring (sP) and alcohol‐nonpreferring (sNP) rats; (ii) the AVP mRNA levels are altered by long‐term alcohol drinking in sP rats; and (iii) the V1b receptor antagonist SSR149415 alters alcohol drinking in sP rats. Methods: In Experiment 1, AVP mRNA levels were measured in the Me/CeA and MH of alcohol‐naïve sP and sNP rats, and sP rats exposed to the standard, homecage 2‐bottle “alcohol versus water” choice regimen 24 h/d for 17 days. In Experiment 2, SSR149415 (0, 3, 10, or 30 mg/kg; intraperitoneal) was acutely administered 30 minutes before lights off to alcohol‐experienced sP rats. Alcohol, water, and food intake were monitored 6 and 24 hours later. Results: We found higher basal AVP mRNA levels in both Me/CeA and MH of alcohol‐naïve sP than sNP rats; alcohol consumption decreased AVP mRNA levels in both brain regions of sP rats, suggesting genetically determined differences between the 2 rat lines and in the effects of alcohol drinking in sP rats. Acute treatment with SSR149415 significantly reduced alcohol intake of sP rats. Conclusion: The stress‐responsive AVP/V1b receptor system is 1 component of the neural circuitry underlying high alcohol drinking in sP rats.