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: 1. 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. 2. 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. 3. post-intervention, which will consist in the re-assessment of the variables previously assessed in the pre-intervention phase. 4. follow-up, consisting of the assessment and monitoring of levels of alcohol consumption and craving during the 3 months following the intervention phase.
Study Type
INTERVENTIONAL
Allocation
RANDOMIZED
Purpose
PREVENTION
Masking
TRIPLE
Enrollment
82
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).
Psychological Neuroscience Laboratory, Psychology Research Center, University of Minho
Braga, Gualtar, Braga, Portugal
RECRUITINGFecal Microbiota - Species Richness
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.
Time frame: At baseline (pre-intervention)
Fecal Microbiota - Species Richness
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.
Time frame: Immediately post-intervention.
Fecal Microbiota - Species Diversity
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.
Time frame: At baseline (pre-intervention)
Fecal Microbiota - Species Diversity
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.
Time frame: Immediately post-intervention
Fecal Microbiota - Quantification of SCFAs levels
The concentration of short-chain fatty acids (SCFAs) present in each collected faecal sample shall be quantified by High Performance Liquid Chromatography (HPLC).
Time frame: At baseline (pre-intervention)
Fecal Microbiota - Quantification of SCFAs levels
The concentration of SCFAs present in each collected faecal sample shall be quantified by HPLC.
Time frame: Immediately post-intervention.
Alcohol Consumption - Drinking pattern
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.
Time frame: Immediately post-intervention
Alcohol Consumption - Drinking pattern
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.
Time frame: 3 months post-intervention
Alcohol Craving - Short-term acute 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).
Time frame: Immediately post-intervention
Alcohol Craving - Short-term acute 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).
Time frame: 3 months post-intervention
Neuropsychological Evaluation - Memory
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.
Time frame: At baseline (pre-intervention)
Neuropsychological Evaluation - Memory
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.
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Time frame: Immediately post-intervention
Neuropsychological Evaluation - Emotion and Social Cognition
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.
Time frame: At baseline (pre-intervention)
Neuropsychological Evaluation - Emotion and Social Cognition
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.
Time frame: Immediately post-intervention
Neuropsychological Evaluation - Executive Function
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).
Time frame: At baseline (pre-intervention)
Neuropsychological Evaluation - Executive Function
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).
Time frame: Immediately post-intervention
Alcohol Cue Reactivity - Emotional measures
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.
Time frame: At baseline (pre-intervention)
Alcohol Cue Reactivity - Emotional measures
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.
Time frame: Immediately post-intervention.
Memory Inhibition Performance
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.
Time frame: At baseline (pre-intervention)
Memory Inhibition Performance
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.
Time frame: Immediately post-intervention.
Ability of Emotional Recognition
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.
Time frame: At baseline (pre-intervention)
Ability of Emotional Recognition
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.
Time frame: Immediately post-intervention.
Blood samples - Presence of Inflammatory Markers
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).
Time frame: At baseline (pre-intervention)
Blood samples - Presence of Inflammatory Markers
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).
Time frame: Immediately post-intervention.
Neurofunctional level - Think/No-Think Alcohol (TNTA) Task
The TNTA task assesses memory inhibition mechanisms in alcohol-related contexts. It involves three phases - Learning, Think/No-Think and Memory Test - during which participants memorise pairs of images (human faces with a neutral expression and alcoholic/non-alcoholic beverages) and are subsequently instructed to actively recall or suppress these associations.
Time frame: At baseline (pre-intervention)
Neurofunctional level - Think/No-Think Alcohol (TNTA) Task
The TNTA task assesses memory inhibition mechanisms in alcohol-related contexts. It involves three phases - Learning, Think/No-Think and Memory Test - during which participants memorise pairs of images (human faces with a neutral expression and alcoholic/non-alcoholic beverages) and are subsequently instructed to actively recall or suppress these associations.
Time frame: Immediately post-intervention
Neurofunctional level - Emotional Face Matching (EFM) Task
The EFM task assesses emotional recognition ability and pre-conscious and conscious neural responses to emotional faces and geometric forms. During each trial, participants view three emotional faces or shapes (one on top and two on the bottom, in a triangular configuration) and are instructed to identify the emotion/shape at the top of the screen and match it with the corresponding one at the bottom using handheld response buttons.
Time frame: At baseline (pre-intervention)
Neurofunctional level - Emotional Face Matching (EFM) Task
The EFM task assesses emotional recognition ability and pre-conscious and conscious neural responses to emotional faces and geometric forms. During each trial, participants view three emotional faces or shapes (one on top and two on the bottom, in a triangular configuration) and are instructed to identify the emotion/shape at the top of the screen and match it with the corresponding one at the bottom using handheld response buttons.
Time frame: Immediately post-intervention
Neurofunctional level - Alcohol Cue Reactivity (ACR) Task
The ACR task assesses the emotional and attentional response to alcoholic stimuli. Participants are shown images of alcoholic and non-alcoholic drinks, interspersed with oddball blocks containing neutral objects, to which they must respond by pressing a button. After viewing all images, participants are asked to rate their emotional responses to the alcoholic or non-alcoholic beverage images in terms of valence, arousal, and desire.
Time frame: At baseline (pre-intervention)
Neurofunctional level - Alcohol Cue Reactivity (ACR) Task
The ACR task assesses the emotional and attentional response to alcoholic stimuli. Participants are shown images of alcoholic and non-alcoholic drinks, interspersed with oddball blocks containing neutral objects, to which they must respond by pressing a button. After viewing all images, participants are asked to rate their emotional responses to the alcoholic or non-alcoholic beverage images in terms of valence, arousal, and desire.
Time frame: Immediately post-intervention