Mapping the Impact of Gut Microbiota on Brain and Behavior Through the Lens of GABA

This interventional trial will involve a double-blind placebo-controlled crossover trial structured in 4 appointments: week 0 (W0), week 4 (W4), week 8 (W8) and week 12 (W12). The study will therefore last 12 weeks, and will include a screening visit and 4 study visits.

The interventional study has a cross-over design, and participants will therefore be invited to consume both the active probiotic supplement and the placebo at different times. The probiotic supplement (Lactobacillus brevis) was chosen based on the ability to produce GABA in vitro (Monteagudo et al., 2022). The placebo will be maltodextrin The products will be provided as capsules and are identical in texture and appearance.

For participants to be considered compliant and therefore included in the study they will be require:

1. to take the probiotic supplement or placebo at least 6 out of every 7 days of the trial for the week period; 2. to provide faecal, blood and urine samples and to be tested with a battery involving behaviour and brain measures relating to GABA levels/activity and 3. to complete a food diary using an online app once every 4 weeks.

As age and BMI are factors associated with significant variation in gut microbiota composition/brain and behaviour functioning, both study arms will be matched on these criteria.

All interested Individuals will be invited to complete an initial screening questionnaire, which includes questions related to a) the study inclusion criteria. Individuals who meet the study inclusion criteria will then be asked to complete b) an MRI contraindications questionnaire and, in absence of any MRI-related contraindications, they will be asked to c) complete a medical/lifestyle questionnaire to confirm the absence of any exclusion criteria.

Individuals who meet the inclusion criteria will be invited to attend a screening visit at the University of Reading for familiarisation with the procedures and for any clarification they might need. Participants will be provided with a sample collection kit (for faecal and urine sample collection), will be instructed on how to use it, and will be asked to track their diet using an online app (eNutri).

All study appointments will take place at the University of Reading. On the day of the study appointment, participants will be asked to provide a fresh faecal sample and spot urine sample (first morning urine), collected using the sample kit they were provided with at the screening visit. A venous blood sample (equal to 10ml/ 2tsp) will be collected by experienced and trained phlebotomists at the University of Reading. Participants will then undergo a neuroimaging battery, which will include structural magnetic resonance images, magnetic resonance spectroscopy (MRS) to quantify concentrations of GABA in determined regions of the brain, and functional magnetic resonance imaging to evaluate functional connectivity between brain areas. Participants will then complete a battery of GABA-dependent behavioural assays including a facial emotion recognition task, a motor learning task, and a tactile frequency discrimination task. Participants will then complete the Autism Spectrum Quotient (AQ), the State and Trait Anxiety Inventory (STAI), and the Centre for Epidemiological Studies Depression Scale (CES-D). Participants will complete this battery of tasks at each of the four study visits at W0, W4, W8 and W12.

At the end of visit 1 (W0), participants will be provided with either the probiotic OR placebo supplement to consume daily for the next 4 weeks, i.e. between study visits 1 and 2. At the end of visit 2 (W4), participants will not be provided with any supplements as they will enter their 4-week period of washout. At the end of visit 3 (W8), participants will be provided with either the probiotic OR placebo supplement to consume daily for the next 4 weeks. If the participant received the probiotic following visit 1, they will receive the placebo at this point, and vice versa. Participants will be asked to take this daily until their fourth and final study visit (W12). Study visit 4 finalises the study.

• Changes in faecal bacteria quantity assessed by Fluorescent In Situ Hybridisation Followed by Flow Cytometry (FISH-FCM) [Срок оценки: 1. Baseline (Week 0), 2. Following intervention (Week 4 or Week 12, depending on intervention arm assignment), 3. Following washout (Week 8), 4. Following placebo (Week 4 or Week 12, depending on intervention arm assignment)]
• Changes in compositional diversity of faecal microbiota attributable to intervention assessed by 16S ribosomal RNA (16S rRNA) gene amplicon sequencing [Срок оценки: 1. Baseline (Week 0), 2. Following intervention (Week 4 or Week 12, depending on intervention arm assignment)]
• Changes in metabolic profile in faecal samples assessed by Nuclear Magnetic Resonance spectroscopy (NMR) [Срок оценки: 1. Baseline (Week 0), 2. Following intervention (Week 4 or Week 12, depending on intervention arm assignment), 3. Following washout (Week 8), 4. Following placebo (Week 4 or Week 12, depending on intervention arm assignment)]
• Changes in metabolic profile in urine samples assessed by Nuclear Magnetic Resonance spectroscopy (NMR) [Срок оценки: 1. Baseline (Week 0), 2. Following intervention (Week 4 or Week 12, depending on intervention arm assignment), 3. Following washout (Week 8), 4. Following placebo (Week 4 or Week 12, depending on intervention arm assignment)]
• Changes in metabolic profile in serum samples assessed by Nuclear Magnetic Resonance spectroscopy (NMR) [Срок оценки: 1. Baseline (Week 0), 2. Following intervention (Week 4 or Week 12, depending on intervention arm assignment), 3. Following washout (Week 8), 4. Following placebo (Week 4 or Week 12, depending on intervention arm assignment)]
• Changes in blood pressure assessed by measures of systolic and diastolic blood pressure [Срок оценки: 1. Baseline (Week 0), 2. Following intervention (Week 4 or Week 12, depending on intervention arm assignment), 3. Following washout (Week 8), 4. Following placebo (Week 4 or Week 12, depending on intervention arm assignment)]
• Changes in motor function performance assessed by reaction time [Срок оценки: 1. Baseline (Week 0), 2. Following intervention (Week 4 or Week 12, depending on intervention arm assignment), 3. Following washout (Week 8), 4. Following placebo (Week 4 or Week 12, depending on intervention arm assignment)]
• Changes in emotion recognition performance assessed by reaction time and accuracy [Срок оценки: 1. Baseline (Week 0), 2. Following intervention (Week 4 or Week 12, depending on intervention arm assignment), 3. Following washout (Week 8), 4. Following placebo (Week 4 or Week 12, depending on intervention arm assignment)]
• Changes in tactile discrimination performance assessed by reaction time, static threshold and dynamic threshold [Срок оценки: 1. Baseline (Week 0), 2. Following intervention (Week 4 or Week 12, depending on intervention arm assignment), 3. Following washout (Week 8), 4. Following placebo (Week 4 or Week 12, depending on intervention arm assignment)]
• Changes in levels of trait and state anxiety assessed by the State-Trait Anxiety Inventory (STAI) [Срок оценки: 1. Baseline (Week 0), 2. Following intervention (Week 4 or Week 12, depending on intervention arm assignment), 3. Following washout (Week 8), 4. Following placebo (Week 4 or Week 12, depending on intervention arm assignment)]
• Changes in levels of depression assessed by the Centre for Epidemiological Studies Depression questionnaire (CES-D) [Срок оценки: 1. Baseline (Week 0), 2. Following intervention (Week 4 or Week 12, depending on intervention arm assignment), 3. Following washout (Week 8), 4. Following placebo (Week 4 or Week 12, depending on intervention arm assignment)]