P. De Witte

Ethanol and amino acids in the central nervous system: assessment of the pharmacological actions of acamprosate.

Ethanol induces alterations in the central nervous system by differentially interfering with a number of neurotransmitter systems, although the mechanisms by which such effects are executed are not well understood. The present review therefore, is designed to ascertain the effect of ethanol on both excitatory and inhibitory amino acid neurotransmitters, as well as the sulphonated amino acid taurine, assayed by the microdialysis technique within specific brain regions of rat during different types of alcohol intoxication, acute and chronic, as well as during the withdrawal period. Such an understanding of these pharmacological actions of ethanol on neurotransmitters is essential in order to provide the impetus for the development of appropriate therapeutic intervention to ameliorate the multitude of neurochemical disorders induced by ethanol. In addition the possible mode of action of a new therapeutic drug for the treatment of alcoholism, acamprosate will be discussed. The first part of this review will be limited to studies of the effect of ethanol on both amino acid neurotransmitters and the sulphonated amino acid taurine, a possible neuromodulator. While, the second part will seek to establish the possible mechanism of action of a new therapeutic drug, acamprosate, which is used to combat the effects of ethanol, particularly during the craving period, as well as maintaining abstinence in weaned alcoholics.

Naloxone Reduces Alcohol Intake in a Free-choice Procedure Even When Both Drinking Bottles Contain Saccharin Sodium Or Quinine Substances

Male Wistar rats were able to drink water and different percentages of ethyl alcohol in a free-choice paradigm. Our results showed a clear-cut preference for 2 and 3% ethanol concentration. Addition of quinine in both drinking bottles increased preference for alcohol, while dilution of saccharin in both bottles totally suppressed this preference for the alcohol bottle. In this alcohol-water choice procedure, naloxone decreased the amount of alcohol intake without altering the amount drunk from the water bottle. This decrease was independent of the palatable or nonpalatable substances added to alcohol and also independent of the percentage of alcohol. Interpretation was done in terms of action of naloxone on the rewarding property of alcohol, or in terms of satiety mechanism for alcohol regulated by naloxone.

Ethanol but not acetaldehyde induced changes in brain taurine: a microdialysis study

Summary.Research has suggested that catalase plays a role in mediating ethanol’s psychopharmacological effects. Catalase is an enzyme that oxidizes ethanol to acetaldehyde. It has been reported that when catalase activity is reduced by 3-amino-1,2,4-triazole (AT), rats reduce their intake and preference for ethanol. The present study assessed the effects of AT on the brain amino acids levels following ethanol administration in Wistar rats. The study consisted of three parts. In the first part, we found no effects of acute and chronic intraperitoneally administered acetaldehyde on amino acids dialysate levels in nucleus accumbens. In the second part, AT was administered five hours prior to ethanol or its vehicle. Ethanol significantly affected the levels of taurine in rat pre-treated with AT. In the final part, ethanol was administered following the pre-treatment with AT but the dependent variable was the concentration of ethanol in the brain.

Effects of naloxone on the self-stimulation behavior of the postero-lateral area of the hypothalamus in rats — Influence of procedural conditions

Rats exhibiting self-stimulation behavior through chronic electrodes implanted in the posterolateral part of the hypothalamus were subcutaneously injected with low doses (0.003–0.3 mg/kg) of naloxone. The animals were allowed to self-regulate the duration of rewarding brain stimulation. It was found that naloxone increases the duration of self-stimulation in rats in which the brain stimulation had previously been associated with footshock. Vehicle injections or injections of naloxone in rats that had not received footshock prior to testing, did not modify self-stimulation behavior. It is suggested that naloxone may facilitate an aversive central component of the brain stimulation; the conditioned rats therefore increased the duration of brain stimulation to compensate for this negative process.

O6FREE ORAL COMMUNICATIONS 6: PSYCHOSOCIAL FACTORS AND ALCOHOL USE DISORDERSO6.1SEXUAL ORIENTATION AND DRUG OF CHOICEO6.2SUSCEPTIBILITY OF AGE AND GENDER TO INTERMITTENT ALCOHOL ABUSEO6.3HIGH EDUCATIONAL LEVELS AS PROTECTIVE FACTOR AGAINST DRUG AND ALCOHOL CONSUMPTIONO6.4AGE OF ONSET, PHYSICAL AND MENTAL HEALTH AND SOCIAL FUNCTIONING IN OLDER ALCOHOL-DEPENDENT INPATIENTS

# O6.1 SEXUAL ORIENTATION AND DRUG OF CHOICE {#article-title-2} There has been some previous report on an association between sexual orientation and substance use, most studies reporting on elevated rates of illegal drug use among LGBs. Despite numerous assertions on supposed drugs of choice among gay/bisexual men (poppers, metamphetamines etc.), the hypothesis has, to our knowledge, not been specifically tested in a population study. Methods. Data from a representative sample of Swiss men (n = 5,387) approximately 20 years old were collected between August 2010 and November 2011. 167(2.8) reported to be gay/bisexual. Results. While no differences could be found regarding lifetime consumption of alcohol, tobacco and cannabis, gay/bisexual men had, compared to heterosexual participants, more often tried other illegal substances (heroin, cocaine, stimulants, hallucinogens etc.). No differences were found regarding the 12-month prevalence among the ever users, with the exception of poppers, which consumption gay/bisexual men had continued more often. Conclusion. Gay/bisexual men do not differ from heterosexual men regarding the trying out of drugs, but differed with regard to the maintenance of poppers consumption. # O6.2 SUSCEPTIBILITY OF AGE AND GENDER TO INTERMITTENT ALCOHOL ABUSE {#article-title-3} Intermittent alcohol abuse (binge drinking) may induce brain damage as well as cognitive impairment in adolescents. However it is unknown whether such alcohol abuse will also have such a devastating effect in adults as well as pre-adolescents engaged in under age drinking. In these present studies, an intermittent ethanol regimen (2or3g/kg, 3x/day for 2 days, followed by 5 days of abstinence for a total of three weeks) was administered to male and female pre-adolescent, adolescent and adult rats. Microdialysis studies identified significant changes in glutamate and arginine basal levels in the nucleus accumbens, NAc, of a) pre-adolescent male and females (glutamate increased; arginine decreased), b) adolescent male (glutamate decreased; arginine increased) and female (glutamate increased; arginine increased or decreased respectively for low and high ethanol dose) and, c) adult male (glutamate decreased; arginine increased) and female (glutamate increased; arginine decreased) rats after 3 weeks of the ethanol dosing regimen. In addition the concentrations of these two amino acids varied between the different groups. A further challenge with ethanol induced variable effects in the release of glutamate and arginine in each of these groups. Significant losses of hippocampal neuronal cells were evident in each of the groups, with the female adolescent rats showing the highest cell loss. These studies indicate that female adolescent rats are more susceptible to the toxic effects of binge drinking, while the male pre-adolescent, adolescent and adult rats appeared to be more resilient. # O6.3 HIGH EDUCATIONAL LEVELS AS PROTECTIVE FACTOR AGAINST DRUG AND ALCOHOL CONSUMPTION {#article-title-4} Background & Aims. Alcohol consumption is linked to social-cultural aspects in Northeast Italy. Precedently alcohol was consumed daily by all members of the family in Northern Italy (steady drinking). This study aimed to determine the prevalence of alcohol and drug consumption in drivers on weekend nights. Methods. As part of the government sponsored “Safe Roads” Program (November 2008 to September 2012) 3150 drivers were stopped in Padova (Italy) by law enforcement officials between midnight and 6. All underwent a preliminary alcohol test and if positive also a urine test for drugs. T test and multivariate analysis were used. Results. 1068 (909M-85% and 159F-15%; mean age 30.5 yr) drivers underwent the Breathalyzer test. 714 of 3150 (22.6%) were found positive (mean alcohol 0.77g/l; range 0-2.67). 242 of 1068 (22.6%) were also positive for drugs. Drug use was more frequent in those resulting positive to alcohol (23,6% vs 15,4%; p = 0.006209). The highest mean alcohol value (0.88g/l) was found between 4 and 5 am (p = 0.006509). Positivity rate for drugs and mean blood alcohol content resulted higher in subjects with lower educational levels (30,8%vs18,0%, p < 0,001 and 0,689g/l vs 1,027, p = 0,00782). At the multivariate analysis drug use is correlated with age, sex, scholar level and alcohol, instead positive test for alcohol is correlated only with scholar level and use of drugs. Conclusions. The prevalence of drugs and alcohol consumption on weekends is high. High educational levels may be protective against the use of drugs and binge drinking. # O6.4 AGE OF ONSET, PHYSICAL AND MENTAL HEALTH AND SOCIAL FUNCTIONING IN OLDER ALCOHOL-DEPENDENT INPATIENTS {#article-title-5} Background. Alcohol dependence is a severe and often chronic condition with a strong impact on physical and mental health and social functioning, especially in older patients. This study aims to determine whether older alcohol-dependent inpatients with early (age <25), late (25-44), and very late (≥45) onset of alcohol dependence differ in physical and mental health and social functioning. Methods. In a specialized inpatient detoxification ward for older patients in The Hague, The Netherlands, the Addiction Severity Index was administered to 157 alcohol-dependent patients aged 50 and over (38% women, mean age 62.7 ± 6.5). Univariate and multivariate analyses were conducted to examine the association between early, late, and very late age of onset of alcohol dependence and indicators of physical and mental health and social functioning. Results. As a group, older alcohol-dependent patients had substantial physical, mental and social problems, which were largely independent of the age of onset of alcohol dependence. Patients with early onset alcohol dependence had more chronic somatic symptoms (OR = 3.08, 95% CI 1.19-7.97, p = 0.02) and more suicidal thoughts (OR = 6.60, 95% CI 1.31-33.3, p = 0.02) than patients with late onset alcohol dependence. The very late onset group did not significantly differ from the other two groups in any of the variables under study. Conclusions. Despite previous studies showing more favorable outcomes for the (very) late onset compared to the early onset alcohol-dependent group, their (comorbid) mental health and social problems are in many respects similar and should not be underestimated.

S06ROLE OF INNATE IMMUNE SYSTEM IN THE ETHANOL-INDUCED BRAIN DAMAGE, BEHAVIOURAL DYSFUNCTIONS AND ADDICTIONS06.1BINGE DRINKING INDUCES SIGNIFICANT CHANGES IN THE INNATE IMMUNE SYSTEMS06.2THE NF-KAPPAB SYSTEM IN TRANSCRIPTIONAL CONTROL OF MALADAPTIVE AND PROTECTIVE RESPONSES IN HUMAN ALCOHOLICSS06.3ROLE OF THE TLR4 RESPONSE IN THE NEUROINFLAMMATION, BRAIN DAMAGE AND BEHAVIOURAL DYSFUNCTIONS INDUCED BY CHRONIC ETHANOL CONSUMPTIONS06.4INDUCTION OF INNATE IMMUNE GENES BY ETHANOL CREATES THE NEUROBIOLOGY OF ADDICTION

Chronic and intermittent alcohol consumption leads to cognitive impairment in the brain due to the ethanols action on specific neurotransmitter systems and intricate signalling pathways. Glial cells actively participate in brain function by nurturing neurons and facilitating neuronal activity as well having an immunological role to protect brain cells from invading pathogens. Dysregulation of this immune function induced by intermittent alcohol abuse may shift the homeostatic balance of inflammatory mediators to a proinflammatory state, activating microglia and inducing neuronal loss thereby inducing behavioural and cognitive impairments. Molecules, which could prevent the microglial activation and cytokine production would downregulate the pro-inflammatory state and help to prevent the decline of cognitive impairment. Studies of a ‘binge drinking’ adolescent female rats identified increased levels of glutamate in the dentate gyrus region of the hippocampus, which were associated with activated microglia. Such microglia will release glutamate when activated as well as a plethora of pro-inflammatory cytokines, e.g. IL-6 and TNFα. Oral administration of a taurine analogue, ethane-β-sultam to such ‘binge drinking’ rats for a 3-week period stabilized IKBα within the microglial cell, thereby preventing NFkappaB translocation to the nucleus and cytokine production. Activated microglia were no longer visible after immunohistochemical staining of the dentate gyrus brain region. The innate immune system, which is activated by intermittent alcohol use, can be suppressed by the use of molecules which target specific activators of this system, i.e. NFkappaB. ERAB funding is gratefully acknowledged. # S06.2 THE NF-KAPPAB SYSTEM IN TRANSCRIPTIONAL CONTROL OF MALADAPTIVE AND PROTECTIVE RESPONSES IN HUMAN ALCOHOLICS {#article-title-2} Alcohol dependence and associated cognitive impairment appear to result from maladaptive neuroplasticity in response to chronic alcohol consumption, neuroinflammation and neurodegeneration. The inherent stability of behavioral alterations associated with the addicted state suggests that transcriptional and epigenetic mechanisms are operative. NF-kappaB transcription factors are regulators of synaptic plasticity and inflammation, and responsive to a variety of stimuli including alcohol. These factors are abundant in the brain where they have diverse functions that depend on the composition of the NF-kappaB complex and cellular context. In neuron cell bodies, NF-kappaB is constitutively active, and involved in neuronal injury and neuroprotection. However, at the synapse, NF-kappaB is present in a latent form and upon activation is transported to the cell nucleus. In glia, NF-kappaB is inducible and regulates inflammatory processes that exacerbate alcohol-induced neurodegeneration. Animal studies demonstrate that acute alcohol exposure transiently activates NF-kappaB, which induces neuroinflammatory responses and neurodegeneration. Our postmortem studies of brains of human alcoholics suggest that cycles of alcohol consumption/withdrawal cause adaptive changes in the NF-kappaB system that permit the system to better tolerate excessive stimulation. This tolerance, ensuring a low degree of responsiveness to applied stimuli, may represent a compensatory response that protects brain cells against alcohol neurotoxicity. This is supported by findings showing preferential downregulation of pro-apoptotic gene expression in the affected brain areas in human alcoholics. Although further verification is needed, we speculate that NF-kappaB-driven neuroinflammation and disruption to neuroplasticity play a significant role in regulating alcohol dependence and cognitive impairment. # S06.3 ROLE OF THE TLR4 RESPONSE IN THE NEUROINFLAMMATION, BRAIN DAMAGE AND BEHAVIOURAL DYSFUNCTIONS INDUCED BY CHRONIC ETHANOL CONSUMPTION {#article-title-3} Toll-like receptors (TLRs) play an important role in the innate immune response, and emerging evidence indicates their role in brain injury and neurodegeneration. Our recent results have demonstrated that ethanol is capable of activating glial TLR4 receptors and that the elimination of these receptors in mice protects against ethanol-induced glial activation, induction of inflammatory mediators and apoptosis. However, whether ethanol-induced inflammatory damage causes behavioural and cognitive consequences, and if behavioural alterations are dependent of TLR4 functions are presently unknown. Here we show in mice drinking alcohol for 5 months, followed by a 15-day withdrawal period, that activation of the astroglial and microglial cells in frontal cortex and striatum is maintained and that these events are associated with cognitive and anxiety-related behavioural impairments in wild-type (WT) mice, as demonstrated by testing the animals with object memory recognition, conditioned taste aversion and dark and light box anxiety tasks. Mice lacking TLR4 receptors (TLR4−/−) are protected against ethanol-induced inflammatory damage, and behavioural associated effects. We further assess the possibility of the epigenetic modifications participating in short- or long-term behavioural effects associated with neuroinflammatory damage. We show that chronic alcohol treatment decreases H4 histone acetylation and histone acetyltransferases activity in frontal cortex, striatum and hippocampus of WT mice. Alterations in chromatin structure were not observed in TLR4−/− mice. These results provide the first evidence of the role that TLR4 functions play in the behavioural consequences of alcohol-induced inflammatory damage and suggest that the epigenetic modifications mediated by TLR4 could contribute to short- or long-term alcohol-induced behavioural or cognitive dysfunctions. This study was supported by SAF-2009-07503, Institute of Heath, Carlos III (RTA-Network). # S06.4 INDUCTION OF INNATE IMMUNE GENES BY ETHANOL CREATES THE NEUROBIOLOGY OF ADDICTION {#article-title-4} Addiction evolves through progressively reduced behavioral control and cognitive flexibility with increasing negative emotion and craving. Recent discoveries indicating neuroimmune signaling contribute to addiction and co-morbid depression. Low threshold microglia undergoes progressive stages of innate immune activation involving astrocytes and neurons with repeated drug abuse, stress and immune signals. Increased brain NF-κB transcription of proinflammatory chemokines, cytokines, oxidases, proteases, TLR and other genes create loops amplifying NF-κB transcription and neuroimmune gene expression. Human post-mortem alcoholic brain has increased microglial markers, chemokine-MCP1, TLR receptors and endogenous TLR agonists. Ethanol activates persistent neuroimmune signaling through the formation of loops of NF-κB transcription in glia contributing to a hyperglutamate state. Chronic ethanol treatment induces reversal learning deficits coincident with frontal cortical damage mimicking human drug addict deficits in behavioral flexibility. Increasing limbic negative emotion and depression-like behavior are reflected in hippocampal neurogenesis. Chronic ethanol inhibits neurogenesis coincident with depression-like behavior with both reversed by antidepressants. Antidepressants, naltrexone and anti-inflammatory drugs block ethanol neuroimmune activation, inhibition of neurogenesis and neurotoxicity. The hypothesis that neuroimmune gene induction underlies addiction and affective disorders creates new targets for therapy. This study was supported by NIH and NIAAA.