The Brain, the Bug, and the Binge: the Interplay Between Binge Drinking, Gut Microbiota, and Brain Functioning

The Brain, the Bug, and the Binge: the Interplay Between Binge Drinking, Gut Microbiota, and Brain Functioning

The Brain, the Bug, and the Binge: a Double-blind, Randomized Controlled Trial Investigating the Interplay Between Binge Drinking, Gut Microbiota and Brain Functioning

Adolescence and youth are periods of significant maturational changes which seems to involve greater susceptibility to disruptive events in the brain such as binge drinking (BD). This prevalent pattern of consumption -characterized by repeated alcohol intoxications- is of special concern, as it has been associated with major neurocognitive impairments in the young brain. Recent studies indicate that alcohol may disrupt the gut microbiota (GM) and that these disruptions may lead to impairments in brain and behavior. Also, interventions with psychobiotics have been shown to result in reductions in alcohol-induced damage and in improvements on cognitive and brain functioning. Thus, the present proposal will explore the effects of BD on GM. Additionally, a GM intervention with psychobiotics both in-vivo and in-vitro, will determine whether improvements in GM composition/function may lead to reductions of alcohol-induced brain damage in BD-population, a barely unexplored research field with major clinical applications.

The present study protocol aims to determine the interaction between alcohol consumption, brain function and gut microbiota through several levels of analysis, including techniques to measure brain activity (i.e., magnetic resonance imaging), paradigms to measure cognitive performance, collection of stool and blood samples, and questionnaires. Additionally, this study will investigate the relationship between alcohol, brain activity and gut microbiota and how this can be modified through our diet. The sample will be composed by a cohort of young college students (18-23 years) from the University of Minho (UM; Braga, Portugal) selected according to their drinking patterns. Eighty-two participants will be recruited from UM: 36 non/low-drinkers and 46 binge drinkers (BDs) matched for age and gender. Recruitment will be carried out through an online survey broadcasted using the institutional email. This survey will include a simple sociodemographic section and items regarding the use of alcohol (Alcohol Use Disorder Identification Test - AUDIT, frequency of alcohol consumption, number of drinks consumed on each day of the past week, speed of drinking, etc.). After sample selection, participants will be submitted to the following steps: (1) clinical interview - addresses questions relating to psychological, medical, personal and family history, including questions related to history of alcohol and drug use and some specific questionnaires relating to substance use, as well as those related to physical and psychological symptoms, and personality; (2) neuroimaging assessment - will consist of a structural and functional magnetic resonance imaging (fMRI) at the Hospital de Guimarães (Portugal), while performing different cognitive tasks; (3) evaluation of some microorganisms residing in the gut and certain inflammatory markers - each participant will be asked to collect stool and blood samples; (4) evaluation of the potential of an intervention with psychobiotics. Thus, this protocol involves the following phases: pre-intervention, consisting of the assessment of the variables of interest to the study by means of a clinical interview, neuropsychological testing, collection of stool and blood samples, and MRI recordings. intervention (only for BDs), consisting of taking a prebiotic for 6 weeks. Depending on the group to which they will be allocated, the participant will take one of two types of fiber: a fiber with benefits for intestinal bacteria (inulin) or a similar fiber with no specific benefits for the intestinal microbiome (maltodextrin). Each participant will not know which group they belong to in order not to bias the results of the study according to scientific standards. post-intervention, which will consist in the re-assessment of the variables previously assessed in the pre-intervention phase. follow-up, consisting of the assessment and monitoring of levels of alcohol consumption and craving during the 3 months following the intervention phase.

Adolescence, Young Adulthood, Alcohol, Binge Drinking, Gut Microbiome, Microbiota-Gut-Brain Axis, Magnetic Resonance Imaging, Pro-inflammatory cytokines, Psychobiotics, Randomized Controlled Trial

Binge Drinkers will be randomly distributed for one of two groups: 23 subjects with inulin intervention and 23 subjects with maltodextrin intervention (~50 % male and ~50 % female in each group). There will also be a non-interventional control group consisting of 36 non/low-drinkers.

For 6 weeks, 23 binge drinkers will be given a daily dose (divided into three times a day) of 15g of a dietary fiber with benefits for intestinal bacteria (inulin).

For 6 weeks, 23 binge drinkers will be given a daily dose (divided into three times a day) of 15g of dietary fiber with no specific benefits for the intestinal microbiome (maltodextrin).

The Alcohol Use Disorder Identification Test (AUDIT) will be administered to characterize the drinking pattern of the participants. AUDIT scores ≤ 4 reveal low risk of alcohol use; scores between 5 and 20 represent excessive alcohol consumption; and scores ≥ 20 indicate very high risk for alcohol dependence and warrant further diagnostic evaluation for alcohol dependence.

The AUDIT will be administered to characterize the drinking pattern of the participants. AUDIT scores ≤ 4 reveal low risk of alcohol use; scores between 5 and 20 represent excessive alcohol consumption; and scores ≥ 20 indicate very high risk for alcohol dependence and warrant further diagnostic evaluation for alcohol dependence.

The AUDIT will be administered to characterize the drinking pattern of the participants. AUDIT scores ≤ 4 reveal low risk of alcohol use; scores between 5 and 20 represent excessive alcohol consumption; and scores ≥ 20 indicate very high risk for alcohol dependence and warrant further diagnostic evaluation for alcohol dependence.

The AUDIT will be administered to characterize the drinking pattern of the participants. AUDIT scores ≤ 4 reveal low risk of alcohol use; scores between 5 and 20 represent excessive alcohol consumption; and scores ≥ 20 indicate very high risk for alcohol dependence and warrant further diagnostic evaluation for alcohol dependence.

Short-term alcohol craving levels will be assessed using the Alcohol Craving Questionnaire - Short form Revised (ACQ-SF-R) at the present moment. Total minimum score: 1 (low level of alcohol craving); Total maximum score: 7 (high level of alcohol craving).

Short-term alcohol craving levels will be assessed using the Alcohol Craving Questionnaire - Short form Revised (ACQ-SF-R) at the present moment. Total minimum score: 1 (low level of alcohol craving); Total maximum score: 7 (high level of alcohol craving).

Short-term alcohol craving levels will be assessed using the ACQ-SF-R at the present moment. Total minimum score: 1 (low level of alcohol craving); Total maximum score: 7 (high level of alcohol craving).

Short-term alcohol craving levels will be assessed using the ACQ-SF-R at the present moment. Total minimum score: 1 (low level of alcohol craving); Total maximum score: 7 (high level of alcohol craving).

The use of illicit drugs will be determined by the Drug Use Disorders Identification Test (DUDIT). DUDIT provides information on the level of drug intake and selected criteria for substance abuse/harmful use and dependence according to the ICD-10 and DSM-4 diagnostic systems. In this study, the use of sedative pills, analgesics and tobacco is not an exclusion criterion.

Habitual dietary intake of each participant will be also measured using a Food Frequency Questionnaire. A detailed description, on average, of nutrients and food group values over the past 12 months will be obtained by classifying the frequency of consumption of each food/beverage type on a scale from never or less than once per month to ≥6 per day.

During the intervention phase, each binge drinker should keep a record of everything they ate and drank during three days of each of the 6 weeks: two days a week and one at the weekend, according to their usual practice. They should also record the type of packaging of the food and drink and the place where they consumed them.

The Barratt Impulsiveness Scale (BIS-11) will be used to assess the personality/behavioural construct of impulsiveness including motor (acting without thinking), attentional (an inability to focus attention or concentrate) and non-planning (lack of forethought), in different situations.

The Barratt Impulsiveness Scale (BIS-11) will be used to assess the personality/behavioural construct of impulsiveness including motor (acting without thinking), attentional (an inability to focus attention or concentrate) and non-planning (lack of forethought), in different situations.

The Delayed Matching to Sample (DMS) from Cambridge Neuropsychological Test Automated Battery (CANTAB) will be used to assess both simultaneous visual matching ability and short-term visual recognition memory, for non-verbalisable patterns.

The Delayed Matching to Sample (DMS) from Cambridge Neuropsychological Test Automated Battery (CANTAB) will be used to assess both simultaneous visual matching ability and short-term visual recognition memory, for non-verbalisable patterns.

The Emotion Recognition Task (ERT) from CANTAB will measure the ability to identify six basic emotions (sadness, happiness, fear, anger, disgust, and surprise) in facial expressions along a continuum of expression magnitude.

The Emotion Recognition Task (ERT) from CANTAB will measure the ability to identify six basic emotions (sadness, happiness, fear, anger, disgust, and surprise) in facial expressions along a continuum of expression magnitude.

The performance of the cognitive domain comprising high-level thinking and decision-making will be assessed through CANTAB, namely the Cambridge Gambling Task (CGT, to assess decision-making and risk behaviour outside a learning context), Intra-Extra Dimensional Set Shift (IED, to assess cognitive flexibility), Spatial Working Memory (SWM, to identify working memory strategies and errors) and Stop Signal Task (SST, to measure response inhibition/impulse control).

The performance of the cognitive domain comprising high-level thinking and decision-making will be assessed through CANTAB, namely the Cambridge Gambling Task (CGT, to assess decision-making and risk behaviour outside a learning context), Intra-Extra Dimensional Set Shift (IED, to assess cognitive flexibility), Spatial Working Memory (SWM, to identify working memory strategies and errors) and Stop Signal Task (SST, to measure response inhibition/impulse control).

The reactivity to alcoholic cues will be assessed using the Alcohol Cue Reactivity (ACR) task. The full task includes a total of 80 trials with 40 alcoholic and 40 non-alcoholic images obtained from the Amsterdam Beverage Picture Set. The emotional responses for each image in terms of valence and arousal task, will be registered using the Self-Assessment Manikin (valence: from 1 = "very unpleasant" to 9 ="very pleasant"; arousal: from 1 = "not arousing" to 9 = "highly arousing") during the ACR task.

The reactivity to alcoholic cues will be assessed using the ACR task. The full task includes a total of 80 trials with 40 alcoholic and 40 non-alcoholic images obtained from the Amsterdam Beverage Picture Set. The emotional responses for each image in terms of valence and arousal task, will be registered using the Self-Assessment Manikin (valence: from 1 = "very unpleasant" to 9 ="very pleasant"; arousal: from 1 = "not arousing" to 9 = "highly arousing") during the ACR task.

Memory Inhibition (MI), specifically alcohol-related MI, will be assessed using the Think/No-Think Alcohol (TNTA) task. Percentage of correct responses (for Think, No-Think and Baseline items) in the TNTA task will be computed according to the following formula: ((number of correctly recalled items)/(number of previously learned items))×100. Correct responses correspond to the items learned during the learning phase and correctly recalled during the memory test phase.

MI, specifically alcohol-related MI, will be assessed using the TNTA task. Percentage of correct responses (for Think, No-Think and Baseline items) in the TNTA task will be computed according to the following formula: ((number of correctly recalled items)/(number of previously learned items))×100. Correct responses correspond to the items learned during the learning phase and correctly recalled during the memory test phase.

Emotional recognition capacity will be assessed through the Emotion Discrimination (ED) task. ED assesses the brain's preconscious and conscious responses to emotional faces. The complete task includes a total of 120 images of human faces (60 men and 60 women), showing the main negative emotions: angry, sadness and fear.

Emotional recognition capacity will be assessed through the ED task. ED assesses the brain's preconscious and conscious responses to emotional faces. The complete task includes a total of 120 images of human faces (60 men and 60 women), showing the main negative emotions: angry, sadness and fear.

Faecal samples will be collected from all participants for microbiota α-diversity analysis by 16S rRNA metagenomics (Illumina sequencing). The Chao1 Index will be used as an estimator of nonparametric microbial species richness in each sample.

Faecal samples will be collected only from binge drinkers subjected to the intervention for microbiota α-diversity analysis by 16S rRNA metagenomics (Illumina sequencing). The Chao1 Index will be used as an estimator of nonparametric microbial species richness in each sample.

Faecal samples will be collected from all participants for microbiota α-diversity analysis by 16S rRNA metagenomics (Illumina sequencing). The Shannon Diversity Index (metric combining richness and evenness, with equal weighting given to abundant and rare species) and the Simpson Diversity Index (metric of richness and evenness, in which more weighting is given to abundant species) will be used.

Faecal samples will be collected only from binge drinkers subjected to the intervention for microbiota α-diversity analysis by 16S rRNA metagenomics (Illumina sequencing). The Shannon Diversity Index (metric combining richness and evenness, with equal weighting given to abundant and rare species) and the Simpson Diversity Index (metric of richness and evenness, in which more weighting is given to abundant species) will be used.

The concentration of short-chain fatty acids (SCFAs) present in each collected faecal sample shall be quantified by High Performance Liquid Chromatography (HPLC).

Blood samples will be collected from all participants. The presence and abundance of the following cytokines will be analyzed: Tumour Necrosis Factor α (TNF-α) and Interleukins (IL-1β, IL-6, IL-10).

Blood samples will be collected only from binge drinkers subjected to the intervention. The presence and abundance of the following cytokines will be analyzed: Tumour Necrosis Factor α (TNF-α) and Interleukins (IL-1β, IL-6, IL-10).

Inclusion Criteria: College students whose native language is Portuguese; Age 18-23 years; Binge Drinkers: report (i) drinking 4 (for women)/5 (for men) or more drinks on one occasion at least once a month, (ii) drinking at a speed of at least two drinks per hour during these episodes (which brings blood alcohol concentration to 0.08 g/dL or above), and (iii) having an AUDIT score < 20. Non/Low-Drinkers: report (i) never drinking 4/5 or more drinks on one occasion and (ii) having an AUDIT score ≤ 4. Exclusion Criteria: Use of illicit drugs as determined by the Drug Use Disorders Identification Test (DUDIT); Alcohol abuse (i.e., AUDIT ≥ 20); Personal history of psychopathological disorders (according to DSM-V criteria); History of traumatic brain injury or neurological disorder; Family history (mother/father) of alcoholism diagnosis of substance abuse; Occurrence of one or more episodes of loss of consciousness for more than 30 minutes; Non-corrected sensory deficits; Diagnosis of any gut disease/problems or other medical conditions: inflammatory bowel disease, irritable bowel syndrome, Crohn's Disease, celiac disease, lactose intolerance, autoimmune disease; Consumption of medical drugs with psychoactive effects (e.g., antidepressants, anxiolytics or benzodiazepines) during the 4 weeks prior to the experiment; Use of any of the following drugs in the last 4 weeks: laxatives, antibiotics, anticoagulants, non-steroidal anti-inflammatory drugs, analgesics, corticosteroids; No type of metal object implanted in the body, especially in the head (orthodontic appliances are not excluded).

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