Altered relation between lipopolysaccharide-induced inflammatory response and excitotoxicity in rat organotypic hippocampal slice cultures during ethanol withdrawal.

BACKGROUND: Ethanol (EtOH) causes neurotoxicity by several mechanisms including excitotoxicity and neuroinflammation, but little is known about the interaction between these mechanisms. Because neuroinflammation is known to enhance excitotoxicity, we hypothesized that neuroinflammation contributes to the enhanced excitotoxicity, which is associated with EtOH withdrawal (EWD). The aim of this study was to evaluate the lipopolysaccharide (LPS)-induced inflammatory response of cultured hippocampal tissue during EWD and its effects on the enhanced N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, which occurs at this time. METHODS: Using a neonatal organotypic hippocampal slice culture (OHSC) model, we assessed the effects of NMDA and LPS (separately or combined) during EWD after 10 days of EtOH exposure. Neurotoxicity was assessed using propidium iodide uptake, and the inflammatory response was evaluated by measuring the release of tumor necrosis factor (TNF)-alpha (quantified by enzyme-linked immunosorbent assay) and nitric oxide (NO; quantified by the Griess reaction) into culture media. Furthermore, we explored the potential role of the microglial cell type using immortalized BV2 microglia treated with EtOH for 10 days and challenged with LPS during EWD. RESULTS: As predicted, NMDA-induced toxicity was potentiated by LPS under control conditions. However, during EWD, the reverse was observed and LPS inhibited peak NMDA-induced toxicity. Additionally, LPS-induced release of TNF-alpha and NO during EWD was reduced compared to control conditions. In BV2 microglia, following EtOH exposure, LPS-induced release of NO was reduced, whereas TNF-alpha release was potentiated. CONCLUSIONS: During EWD following chronic EtOH exposure, OHSC exhibited a desensitized inflammatory response to LPS and the effects of LPS on NMDA toxicity were reversed. This might be explained by a change in microglia to an anti-inflammatory and neuroprotective phenotype. In support, studies on BV2 microglia indicate that EtOH exposure and EWD do alter the response of these cells to LPS, but this cannot fully explain the changes observed in the OHSC. The data suggest that neuroinflammation and excitotoxicity do interact during EWD. However, the interaction is not as simple as we originally proposed. This in turn illustrates the need to assess the extent, importance, and relation of these mechanisms in models of EtOH exposure producing neurotoxicity.

The Dietary Flavonoid Rhamnetin Inhibits Both Inflammation and Excitotoxicity During Ethanol Withdrawal in Rat Organotypic Hippocampal Slice Cultures.

BACKGROUND: Ethanol (EtOH) causes neurotoxicity via several mechanisms including neuroinflammation (during EtOH exposure), and excitotoxicity (during EtOH withdrawal [EWD]). Alpha7 nicotinic acetylcholine receptor (nAChR) selective agonists have the potential to reduce both. The aim of this study was to evaluate the anti-inflammatory and neuroprotective potential of rhamnetin, a dietary flavonoid with alpha7 nAChR selective activity, in an in vitro model of EtOH-induced neurotoxicity. METHODS: The anti-inflammatory and neuroprotective properties of rhamnetin were assessed in neonatal organotypic hippocampal slice cultures undergoing EWD (or not) and challenged with N-methyl-D-aspartate (NMDA) and/or lipopolysaccharide (LPS). Neurotoxicity was determined using propidium iodide uptake, and the inflammatory response was evaluated by measuring the release of tumor necrosis factor (TNF)-alpha (NO; quantified by ELISA) and nitric oxide (quantified by the Griess reaction) into culture media. RESULTS: As predicted, rhamnetin reduced LPS-induced release of TNF-alpha and NO both under control conditions and during EWD. Additionally, rhamnetin had no effect on NMDA-induced neurotoxicity under control conditions, but significantly reduced NMDA toxicity during EWD. In contrast, rhamnetin had no effect on neurotoxicity induced by NMDA and LPS combined despite reducing TNF-alpha and NO levels under these conditions. CONCLUSIONS: Rhamnetin is anti-inflammatory and neuroprotective during EWD and therefore has potential value in treating neurotoxicity caused by EtOH.

Alcohol conditioned contexts enhance positive subjective alcohol effects and consumption.

Associations between alcohol and the places it is consumed are important at all stages of alcohol abuse and addiction. However, it is not clear how the associations are formed in humans or how they influence drinking, and there are few effective strategies to prevent their pathological effects on alcohol use. We used a human laboratory model to study the effects of alcohol environments on alcohol consumption. Healthy regular binge drinkers completed conditioned place preference (CPP) with 0 vs. 80 mg/100 mL alcohol (Paired Group). Control participants (Unpaired Group) completed sessions without explicit alcohol-room pairings. After conditioning, participants completed alcohol self-administration in either the alcohol- or no alcohol-paired room. Paired group participants reported greater subjective stimulation and euphoria, and consumed more alcohol in the alcohol-paired room in comparison to the no alcohol-paired room, and controls tested in either room. Moreover, the strength of conditioning significantly predicted drinking; participants who exhibited the strongest CPP consumed the most alcohol in the alcohol-paired room. This is the first empirical evidence that laboratory-conditioned alcohol environments directly influence drinking. The results also confirm the viability of the model to examine the mechanisms by which alcohol environments stimulate drinking and to test strategies to counteract their influence on behavior.

Test-retest reliability of the underlying latent factor structure of alcohol subjective response.

RATIONALE: Alcohol subjective experiences are multi-dimensional and demonstrate wide inter-individual variability. Recent efforts have sought to establish a clearer understanding of subjective alcohol responses by identifying core constructs derived from multiple measurement instruments. OBJECTIVE: The aim of this study was to evaluate the temporal stability of this approach to conceptualizing alcohol subjective experiences across successive alcohol administrations in the same individuals. METHODS: Healthy moderate alcohol drinkers (n = 104) completed six experimental sessions each, three with alcohol (0.8 g/kg), and three with a non-alcoholic control beverage. Participants reported subjective mood and drug effects using standardized questionnaires before and at repeated times after beverage consumption. We explored the underlying latent structure of subjective responses for all alcohol administrations using exploratory factor analysis and then tested measurement invariance over the three successive administrations using multi-group confirmatory factor analyses. RESULTS: Exploratory factor analyses on responses to alcohol across all administrations yielded four factors representing "Positive mood," "Sedation," "Stimulation/Euphoria," and "Drug effects and Urges." A confirmatory factor analysis on the separate administrations indicated acceptable configural and metric invariance and moderate scalar invariance. CONCLUSIONS: In this study, we demonstrate temporal stability of the underlying constructs of subjective alcohol responses derived from factor analysis. These findings strengthen the utility of this approach to conceptualizing subjective alcohol responses especially for use in prospective and longitudinal alcohol challenge studies relating subjective response to alcohol use disorder risk.

Dose-related effects of delta-9-THC on emotional responses to acute psychosocial stress.

OBJECTIVES: Cannabis smokers often report that they use the drug to relax or to relieve emotional stress. However, few clinical studies have shown evidence of the stress-relieving effects of cannabis or cannabinoid agonists. In this study, we sought to assess the influence of delta-9-tetrahydrocannabinol (THC), a main active ingredient of cannabis, upon emotional responses to an acute psychosocial stressor among healthy young adults. METHODS: Healthy volunteers (N=42) participated in two experimental sessions, one with psychosocial stress (Trier Social Stress Test, TSST) and another with a non-stressful task, after receiving 0 (N=13), 7.5mg (N=14) or 12.5mg (N=15) oral THC. Capsules were administered under randomized, double blind conditions, 2.5h before the tasks began. We measured subjective mood and drug effects, vital signs and salivary cortisol before and at repeated times after the capsule and tasks. Subjects also appraised the tasks, before and after completion. RESULTS: In comparison to placebo, 7.5mg THC significantly reduced self-reported subjective distress after the TSST and attenuated post-task appraisals of the TSST as threatening and challenging. By contrast, 12.5mg THC increased negative mood overall i.e., both before and throughout the tasks, and pre-task ratings of the TSST as threatening and challenging. It also impaired TSST performance and attenuated blood pressure reactivity to the stressor. CONCLUSIONS: Our findings suggest that a low dose of THC produces subjective stress-relieving effects in line with those commonly reported among cannabis users, but that higher doses may non-specifically increase negative mood.

A nicotinic receptor-mediated anti-inflammatory effect of the flavonoid rhamnetin in BV2 microglia.

The alpha7 nicotinic acetylcholine receptor (nAChR) is a potential target in neuroinflammation. Screening a plant extract library identified Solidago nemoralis as containing methyl-quercetin derivatives that are relatively selective ligands for the alpha7 nAChR. Flavonoids are not known for this activity, so we screened a small library of pure flavonoids to confirm our findings. Some flavonoids, e.g. rhamnetin, displaced a selective alpha7 nAChR radioligand from rat brain membranes whereas similar structures e.g. sakuranetin, did not. To evaluate the contribution of this putative nAChR activity to the known anti-inflammatory properties of these flavonoids, we compared their effects on lipopolysaccharide induced release of inflammatory mediators from BV2 microglia. Both rhamnetin and sakuranetin reduced mediator release, but differed in potency (rhamnetin>sakuranetin) and the Hill slope of their concentration-response curves. For rhamnetin the Hill coefficient was >3.0 whereas for sakuranetin the coefficient was 1.0, suggesting that effects of rhamnetin are mediated through more than one mechanism, whereas sakuranetin has a single mechanism. nAChR antagonists decreased the Hill coefficient for rhamnetin toward unity, which suggests that a nAChR-mediated mechanism contributes cooperatively to its overall anti-inflammatory effect. In contrast nAChR antagonists had no effect on the potency or Hill coefficient for sakuranetin, but a concentration of nicotine (1µM) that had no effect alone, significantly increased the Hill coefficient of this flavonoid. In conclusion, the anti-inflammatory effects of rhamnetin benefit cooperatively from a nAChR-mediated mechanism. This action, together with potent free radical scavenging activity, suggests that flavonoids with alpha7 nAChR activity have therapeutic potential in neuroinflammatory conditions.