Gut microbiota signatures of vulnerability to food addiction in mice and humans.

OBJECTIVE: Food addiction is a multifactorial disorder characterised by a loss of control over food intake that may promote obesity and alter gut microbiota composition. We have investigated the potential involvement of the gut microbiota in the mechanisms underlying food addiction. DESIGN: We used the Yale Food Addiction Scale (YFAS) 2.0 criteria to classify extreme food addiction in mouse and human subpopulations to identify gut microbiota signatures associated with vulnerability to this disorder. RESULTS: Both animal and human cohorts showed important similarities in the gut microbiota signatures linked to food addiction. The signatures suggested possible non-beneficial effects of bacteria belonging to the Proteobacteria phylum and potential protective effects of Actinobacteria against the development of food addiction in both cohorts of humans and mice. A decreased relative abundance of the species Blautia wexlerae was observed in addicted humans and of Blautia genus in addicted mice. Administration of the non-digestible carbohydrates, lactulose and rhamnose, known to favour Blautia growth, led to increased relative abundance of Blautia in mice faeces in parallel with dramatic improvements in food addiction. A similar improvement was revealed after oral administration of Blautia wexlerae as a beneficial microbe. CONCLUSION: By understanding the crosstalk between this behavioural alteration and gut microbiota, these findings constitute a step forward to future treatments for food addiction and related eating disorders.

Unraveling the Dynamics of Host-Microbiota Indole Metabolism: An Investigation of Indole, Indolin-2-one, Isatin, and 3-Hydroxyindolin-2-one.

The gut microbiota produces a variety of bioactive molecules that facilitate host-microbiota interaction. Indole and its metabolites are focused as possible biomarkers for various diseases. However, data on indole metabolism and individual metabolites remain limited. Hence, we investigated the metabolism and distribution of indole, indolin-2-one, isatin, and 3-hydroxyindolin-2-one. First, we orally administered a high dose of indole into C57BL/6J mice and measured the concentrations of indole metabolites in the brain, liver, plasma, large and small intestines, and cecum at multiple time points using HPLC/MS. Absorption in 30 min and full metabolization in 6 h were established. Furthermore, indole, indolin-2-one, and 3-hydroxiindolin-2-one, but not isatin, were found in the brain. Second, we confirmed these findings by using stable isotope-carrying indole. Third, we identified 3-hydroxyindolin-2-one as an indole metabolite in vivo by utilizing a 3-hydroxyindolin-2-one-converting enzyme, IifA. Further, we confirmed the ability of orally administered 3-hydroxyindolin-2-one to cross the blood-brain barrier in a dose-dependent manner. Finally, we detected upregulation of the CYP1A2 and CYP2A5 genes, confirming the importance of these cytochrome isoforms in indole metabolism in vivo. Overall, our results provide a basic characterization of indole metabolism in the host and highlight 3-hydroxyindolin-2-one as a potentially brain-affecting indole metabolite.

A Comparison of Methods of Gut Microbiota Transplantation for Preclinical Studies.

The experimental details reported in preclinical fecal microbiota transplantation (FMT) protocols are highly inconsistent, variable, and/or incomplete. We therefore evaluated FMT from a human donor to antibiotic-induced microbial-depleted mice by exploring the effects of six techniques based on antibiotic (AB) or antibiotic + antimycotic (AB + T) gut decontamination, different administration routes, and different dosing intervals on the gut microbial population, assessed using 16S and 18S sequencing. In addition, we explored the effectiveness of FMT in terms of inflammation, physiological, and behavioral outcomes. Our results showed that intrarectal FMT at low dosing intervals better preserved the donor's gut bacterial community at genus level. Furthermore, we showed a lower abundance of several genera of fungi in animals treated with AB + T. In addition, we observed that AB + T gut decontamination followed by per os FMT, once every 3 days, affected behavioral parameters when compared to other FMT techniques. Accordingly, the same FMT groups that showed an association with some of the behavioral tests were also related to specific gut fungal genera, suggesting a possible mediation. Our findings may be useful for optimizing the practice of FMT and also in terms of donor microbiota preservation. This information may help to improve the reproducibility and reliability of FMT studies.

Prebiotics as a Tool for the Prevention and Treatment of Obesity and Diabetes: Classification and Ability to Modulate the Gut Microbiota.

Diabetes and obesity are metabolic diseases that have become alarming conditions in recent decades. Their rate of increase is becoming a growing concern worldwide. Recent studies have established that the composition and dysfunction of the gut microbiota are associated with the development of diabetes. For this reason, strategies such as the use of prebiotics to improve intestinal microbial structure and function have become popular. Consumption of prebiotics for modulating the gut microbiota results in the production of microbial metabolites such as short-chain fatty acids that play essential roles in reducing blood glucose levels, mitigating insulin resistance, reducing inflammation, and promoting the secretion of glucagon-like peptide 1 in the host, and this accounts for the observed remission of metabolic diseases. Prebiotics can be either naturally extracted from non-digestible carbohydrate materials or synthetically produced. In this review, we discussed current findings on how the gut microbiota and microbial metabolites may influence host metabolism to promote health. We provided evidence from various studies that show the ability of prebiotic consumption to alter gut microbial profile, improve gut microbial metabolism and functions, and improve host physiology to alleviate diabetes and obesity. We conclude among other things that the application of systems biology coupled with bioinformatics could be essential in ascertaining the exact mechanisms behind the prebiotic-gut microbe-host interactions required for diabetes and obesity improvement.

Obesity-associated deficits in inhibitory control are phenocopied to mice through gut microbiota changes in one-carbon and aromatic amino acids metabolic pathways.

BACKGROUND: Inhibitory control (IC) is critical to keep long-term goals in everyday life. Bidirectional relationships between IC deficits and obesity are behind unhealthy eating and physical exercise habits. METHODS: We studied gut microbiome composition and functionality, and plasma and faecal metabolomics in association with cognitive tests evaluating inhibitory control (Stroop test) and brain structure in a discovery (n=156), both cross-sectionally and longitudinally, and in an independent replication cohort (n=970). Faecal microbiota transplantation (FMT) in mice evaluated the impact on reversal learning and medial prefrontal cortex (mPFC) transcriptomics. RESULTS: An interplay among IC, brain structure (in humans) and mPFC transcriptomics (in mice), plasma/faecal metabolomics and the gut metagenome was found. Obesity-dependent alterations in one-carbon metabolism, tryptophan and histidine pathways were associated with IC in the two independent cohorts. Bacterial functions linked to one-carbon metabolism (thyX,dut, exodeoxyribonuclease V), and the anterior cingulate cortex volume were associated with IC, cross-sectionally and longitudinally. FMT from individuals with obesity led to alterations in mice reversal learning. In an independent FMT experiment, human donor's bacterial functions related to IC deficits were associated with mPFC expression of one-carbon metabolism-related genes of recipient's mice. CONCLUSION: These results highlight the importance of targeting obesity-related impulsive behaviour through the induction of gut microbiota shifts.

Extinction and reinstatement of an operant responding maintained by food in different models of obesity.

A major problem in treating obesity is the high rate of relapse to abnormal food-taking habits after maintaining an energy balanced diet. Alterations of eating behavior such as compulsive-like behavior and lack of self-control over food intake play a critical role in relapse. In this study, we used an operant paradigm of food-seeking behavior on two different diet-induced obesity models, a free-choice chocolate-mixture diet and a high-fat diet with face validity for a rapid development of obesity or for unhealthy food regularly consumed in our societies. A reduced operant performance and motivation for the hedonic value of palatable chocolate pellets was revealed in both obesity mouse models. However, only mice exposed to high-fat diet showed an increased compulsive-like behavior in the absence of the reinforcer further characterized by impaired operant learning, enhanced impulsivity and intensified inflexibility. We used principal component analysis to globally identify the specific behaviors responsible for the differences among diet groups. Learning impairment and inflexible behaviors contributed to a first principal component, explaining the largest proportion of the variance in the high-fat diet mice phenotype. Reinforcement, impulsion and compulsion were the main contributors to the second principal component explaining the differences in the chocolate-mixture mice behavioral phenotype. These behaviors were not exclusive of chocolate group because some high-fat individuals showed similar values on this component. These data indicate that extended access to hypercaloric diets differentially modifies operant behavior learning, behavioral flexibility, impulsive-like and compulsive-like behavior, and these effects were dependent on the exposure to each specific diet.

Time-course and dynamics of obesity-related behavioral changes induced by energy-dense foods in mice.

Obesity represents an important risk factor contributing to the global burden of disease. The current obesogenic environment with easy access to calorie-dense foods is fueling this obesity epidemic. However, how these foods contribute to the progression of feeding behavior changes that lead to overeating is not well understood and needs systematic assessment. Using novel automated methods for the high-throughput screening of behavior, we here examine mice meal pattern upon long-term exposure to a free-choice chocolate-mixture diet and a high-fat diet with face validity for a rapid development of obesity induced by unhealthy food regularly consumed in our societies. We identified rapid diet-specific behavioral changes after exposure to those high-caloric diets. Mice fed with high-fat chow, showed long-lasting meal pattern disturbances, which initiate with a stable loss of circadian feeding rhythmicity. Mice receiving a chocolate-mixture showed qualitatively similar changes, though less marked, consisting in a transient disruption of the feeding behavior and the circadian feeding rhytmicity. Strikingly, compulsive-like eating behavior is triggered immediately after exposure to both high-fat food and chocolate-mixture diet, well before any changes in body weight could be observed. We propose these changes as behavioral biomarkers of prodromal states of obesity that could allow early intervention.

Microbiota regulation of the Mammalian gut-brain axis.

The realization that the microbiota-gut-brain axis plays a critical role in health and disease has emerged over the past decade. The brain-gut axis is a bidirectional communication system between the central nervous system (CNS) and the gastrointestinal tract. Regulation of the microbiota-brain-gut axis is essential for maintaining homeostasis, including that of the CNS. The routes of this communication are not fully elucidated but include neural, humoral, immune, and metabolic pathways. A number of approaches have been used to interrogate this axis including the use of germ-free animals, probiotic agents, antibiotics, or animals exposed to pathogenic bacterial infections. Together, it is clear that the gut microbiota can be a key regulator of mood, cognition, pain, and obesity. Understanding microbiota-brain interactions is an exciting area of research which may contribute new insights into individual variations in cognition, personality, mood, sleep, and eating behavior, and how they contribute to a range of neuropsychiatric diseases ranging from affective disorders to autism and schizophrenia. Finally, the concept of psychobiotics, bacterial-based interventions with mental health benefit, is also emerging.

Frustrated expected reward induces differential transcriptional changes in the mouse brain.

Frustration represents a particular aspect of the addictive process that is related to loss of control when the expected reward is not obtained. We aim to study the consequences of frustrated expected reward on gene expression in the mouse brain. For this purpose, we used an operant model of frustration using palatable food as reward combined with microarrays. Transcriptomic profiles of frontal cortex, ventral striatum and hippocampus were analysed in five groups of mice: (1) positive control receiving palatable food and the cue light as conditioned stimulus; (2) frustrated group only receiving the cue light; (3) extinction learning group that did not receive palatable food nor the light; (4) negative control that never received the reinforcer nor the light during the whole experiment; and (5) yoked that received palatable food passively. Gene expression changes produced by frustration were revealed in the frontal cortex and ventral striatum, but not in the hippocampus. Most of the changes, such as the modification of the dopamine-DARPP-32 signalling pathway, were common in both areas and estimated to have neuronal origin. Extinction learning induced transcriptional changes only in the ventral striatum, with most genes showing down-regulation and without alteration in the dopamine-DARPP-32 signalling pathway. Active palatable food-seeking behaviour induced changes in gene expression in ventral striatum mainly affecting cell communication. In conclusion, frustration behaviour-induced changes in frontal cortex and ventral striatum mainly related to dopamine-DARPP-32 signalling that could play an important role in the loss of behavioural control during the addictive processes.

Relationships between serotonergic and cannabinoid system in depressive-like behavior: a PET study with [11C]-DASB.

Chronic stress represents a major environmental risk factor for mood disorders in vulnerable individuals. The neurobiological mechanisms underlying these disorders involve serotonergic and endocannabinoid systems. In this study, we have investigated the relationships between these two neurochemical systems in emotional control using genetic and imaging tools. CB1 cannabinoid receptor knockout mice (KO) and wild-type littermates (WT) were exposed to chronic restraint stress. Depressive-like symptoms (anhedonia and helplessness) were produced by chronic stress exposure in WT mice. CB1 KO mice already showed these depressive-like manifestations in non-stress conditions and the same phenotype was observed after chronic restraint stress. Chronic stress similarly impaired long-term memory in both genotypes. In addition, brain levels of serotonin transporter (5-HTT) were assessed using positron emission tomography. Decreased brain 5-HTT levels were revealed in CB1 KO mice under basal conditions, as well as in WT mice after chronic stress. Our results show that chronic restraint stress induced depressive-like behavioral alterations and brain changes in 5-HTT levels similarly to those revealed in CB1 KO mice in non-stressed conditions. These results underline the relevance of chronic environmental stress on serotonergic and endocannabinoid transmission for the development of depressive symptoms. Chronic restraint stress induces depressive-like behavior and reduced 5-HTT levels in WT mice similar to those revealed in non-stressed CB1-KO mice. Reduced 5-HTT in both genotypes increases synaptic 5-HT concentration. The 5-HT release is modulated through CB1 receptors and the absence of inhibitory CB1 receptor causes decreased inhibition of 5-HT release resulting in high synaptic 5-HT concentration that are not further enhanced by stress.

The gut virome is associated with stress-induced changes in behaviour and immune responses in mice.

The microbiota-gut-brain axis has been shown to play an important role in the stress response, but previous work has focused primarily on the role of the bacteriome. The gut virome constitutes a major portion of the microbiome, with bacteriophages having the potential to remodel bacteriome structure and activity. Here we use a mouse model of chronic social stress, and employ 16S rRNA and whole metagenomic sequencing on faecal pellets to determine how the virome is modulated by and contributes to the effects of stress. We found that chronic stress led to behavioural, immune and bacteriome alterations in mice that were associated with changes in the bacteriophage class Caudoviricetes and unassigned viral taxa. To determine whether these changes were causally related to stress-associated behavioural or physiological outcomes, we conducted a faecal virome transplant from mice before stress and autochthonously transferred it to mice undergoing chronic social stress. The transfer of the faecal virome protected against stress-associated behaviour sequelae and restored stress-induced changes in select circulating immune cell populations, cytokine release, bacteriome alterations and gene expression in the amygdala. These data provide evidence that the virome plays a role in the modulation of the microbiota-gut-brain axis during stress, indicating that these viral populations should be considered when designing future microbiome-directed therapies.

Exploration of the association between menopausal symptoms, gastrointestinal symptoms, and perceived stress: survey-based analysis.

OBJECTIVE: The study aimed to evaluate the relationship between menopausal symptoms, gastrointestinal symptoms, and experienced stress in women from premenopause to postmenopause. METHODS: We conducted a cross-sectional study using an anonymous survey that included questions on demographics, health (gynecological, gastrointestinal), and lifestyle (physical activity, sleep, etc) factors, the Perceived Stress Scale (PSS), and the Menopause-Specific Quality of Life Questionnaire (MENQOL). RESULTS: Data of 693 participants aged 50.1 ± 3.2 years were analyzed. We found that the MENQOL total score increased depending on the stages of reproductive aging ( P < 0.001) and positively correlated with PSS scores ( r = 0.47, P < 0.001). Age, reproductive stage, body mass index (BMI), PSS score, diagnosis of depression or anxiety disorder, physical activity, and frequency of defecation appeared to have significant association with the total MENQOL score ( P < 0.05). The analysis within separate MENQOL domains revealed that PSS score and diagnosis of depression or anxiety disorder were associated with higher scores in all MENQOL domains ( P < 0.05) except sexual. Physical activity and the values of the Bristol stool form scale were related to the vasomotor items ( P < 0.05). The frequency of defecation was an independent contributor to the psychosocial and sexual domains ( P < 0.05). BMI, physical activity, and frequency of defecation were associated with physical symptoms ( P < 0.05). CONCLUSIONS: Perceived stress and some gastrointestinal symptoms in women were associated with menopausal symptoms. Reproductive stages, physical activity, BMI, and previously diagnosed depression or anxiety disorder were related to the intensity of menopausal symptoms. However, further research is needed to confirm the relationship between stress, gastrointestinal, and menopausal symptoms.

Microglia isolation from aging mice for cell culture: A beginner's guide.

Microglia, the innate immune cell of the central nervous system, play significant roles in brain development, maintenance, homeostasis, and neuroinflammation. Although numerous methods have been developed to isolate microglia from embryonic or postnatal mouse brains, still major difficulties exist in isolating microglia from adult mice, often resulting in low yield and risk of cellular activation. Therefore, there is a need for a more efficient method to isolate pure and high-yield microglia from adult mice to study various neurodegenerative diseases. The aim of this study was to develop a fully functional protocol for the isolation of microglia by comparing different protocols. We investigated the efficacy of three protocols in terms of cell yield, purity, cellular activation, cellular aging, and migration properties and proposed the modified protocol (PROTOCOL 1), which provides an optimal yield of functional microglial cells with a minimum of material and equipment and allows young researchers with little experience to isolate microglia and helps them to delve deeper into the world of neuroscience.

Corrigendum to: Draft genome sequence dataset of Latilactobacillus curvatus PN39MY isolated from fermented vegetables Data in Brief, 49 (2023) 109436.

[This corrects the article DOI: 10.1016/j.dib.2023.109436.].

Draft genome sequence dataset of Latilactobacillus curvatus PN39MY isolated from fermented vegetables.

Here we report the draft genome sequence of the Latilactobacillus curvatus PN39MY strain. The strain was isolated from Lithuanian traditionally (homemade) fermented cucumber. The genome consisted of 83 contigs with a total size of 1,899,018 bp, an N50 of 40562 and a GC% of 42.1. After sequence trimming, 83 contigs were annotated and 1910 genes were coding sequences. The average nucleotide identity (ANI) between PN39MY and Latilactobacillus curvatus_ZJUNIT8 was 99.45% identifying the strain as Latilactobacillus curvatus. No genes related to antimicrobial resistance or virulence factors were found. The data presented here can be used in comparative genomics to identify antimicrobial resistant genes, plasmids and/or virulence factors that may be present in related Latilactobacillus species. The draft genome sequence data was deposited at NCBI under Bioproject with the accession number PRJNA941180.

In vitro screening and characterization of lactic acid bacteria from Lithuanian fermented food with potential probiotic properties.

The present work aimed to identify probiotic candidates from Lithuanian homemade fermented food samples. A total of 23 lactic acid bacteria were isolated from different fermented food samples. Among these, only 12 showed resistance to low pH, tolerance to pepsin, bile salts, and pancreatin. The 12 strains also exhibited antimicrobial activity against Staphylococcus aureus ATCC 29213, Salmonella Typhimurium ATCC 14028, Streptococcus pyogenes ATCC 12384, Streptococcus pyogenes ATCC 19615, and Klebsiella pneumoniae ATCC 13883. Cell-free supernatants of isolate 3A and 55w showed the strongest antioxidant activity of 26.37 µg/mL and 26.06 µg/mL, respectively. Isolate 11w exhibited the strongest auto-aggregation ability of 79.96% as well as the strongest adhesion to HCT116 colon cells (25.671 ± 0.43%). The selected strains were tested for their synbiotic relation in the presence of a prebiotic. The selected candidates showed high proliferation in the presence of 4% as compared to 2% galactooligosaccharides. Among the strains tested for tryptophan production ability, isolate 11w produced the highest L-tryptophan levels of 16.63 ± 2.25 µm, exhibiting psychobiotic ability in the presence of a prebiotic. The safety of these strains was studied by ascertaining their antibiotic susceptibility, mucin degradation, gelatin hydrolysis, and hemolytic activity. In all, isolates 40C and 11w demonstrated the most desirable probiotic potentials and were identified by 16S RNA and later confirmed by whole genome sequencing as Lacticaseibacillus paracasei 11w, and Lactiplantibacillus plantarum 40C: following with the harboring plasmid investigation. Out of all the 23 selected strains, only Lacticaseibacillus paracasei 11w showed the potential and desirable probiotic properties.

Operant model of frustrated expected reward in mice.

One aspect of the addictive process that has not been thoroughly investigated is the consequence of the frustrated state occurring when the drug is not available. The present study aimed to validate a novel operant model of frustrated expected reward in mice. C57BL/6J mice were trained in operant conditioning maintained by chocolate-flavoured pellets or cocaine. After the completion of high rates of responding on a progressive ratio schedule, the reward was unexpectedly withheld. The consequences of this frustrated behaviour on anxiety, aggressiveness, perseveration, extinction and reinstatement were investigated. Mice exposed to the frustrated event perseverated in the operant responses and showed increased aggressiveness in the resident-intruder test. These animals also showed higher rates of cue-induced reinstatement of drug seeking. The present study provides a reliable operant model in mice to evaluate a frustrated state following reward unavailability. This animal model could be useful to study the behavioural and neurochemical consequences related to the emotional states generated during the omission of a highly expected reward.

miRNA signatures associated with vulnerability to food addiction in mice and humans.

Food addiction is characterized by a loss of behavioral control over food intake and is associated with obesity and other eating disorders. The mechanisms underlying this behavioral disorder are largely unknown. We aimed to investigate the changes in miRNA expression promoted by food addiction in animals and humans and their involvement in the mechanisms underlying the behavioral hallmarks of this disorder. We found sharp similitudes between miRNA signatures in the medial prefrontal cortex (mPFC) of our animal cohort and circulating miRNA levels in our human cohort, which allowed us to identify several miRNAs of potential interest in the development of this disorder. Tough decoy (TuD) inhibition of miRNA-29c-3p in the mouse mPFC promoted persistence of the response and enhanced vulnerability to developing food addiction, whereas miRNA-665-3p inhibition promoted compulsion-like behavior and also enhanced food addiction vulnerability. In contrast, we found that miRNA-137-3p inhibition in the mPFC did not lead to the development of food addiction. Therefore, miRNA-29c-3p and miRNA-665-3p could be acting as protective factors with regard to food addiction. We believe the elucidation of these epigenetic mechanisms will lead to advances toward identifying innovative biomarkers and possible future interventions for food addiction and related disorders based on the strategies now available to modify miRNA activity and expression.

Presence of Blastocystis in gut microbiota is associated with cognitive traits and decreased executive function.

Growing evidence implicates the gut microbiome in cognition. Blastocystis is a common gut single-cell eukaryote parasite frequently detected in humans but its potential involvement in human pathophysiology has been poorly characterized. Here we describe how the presence of Blastocystis in the gut microbiome was associated with deficits in executive function and altered gut bacterial composition in a discovery (n = 114) and replication cohorts (n = 942). We also found that Blastocystis was linked to bacterial functions related to aromatic amino acids metabolism and folate-mediated pyrimidine and one-carbon metabolism. Blastocystis-associated shifts in bacterial functionality translated into the circulating metabolome. Finally, we evaluated the effects of microbiota transplantation. Donor's Blastocystis subtypes led to altered recipient's mice cognitive function and prefrontal cortex gene expression. In summary, Blastocystis warrant further consideration as a novel actor in the gut microbiome-brain axis.

Caudovirales bacteriophages are associated with improved executive function and memory in flies, mice, and humans.

Growing evidence implicates the gut microbiome in cognition. Viruses, the most abundant life entities on the planet, are a commonly overlooked component of the gut virome, dominated by the Caudovirales and Microviridae bacteriophages. Here, we show in a discovery (n = 114) and a validation cohort (n = 942) that subjects with increased Caudovirales and Siphoviridae levels in the gut microbiome had better performance in executive processes and verbal memory. Conversely, increased Microviridae levels were linked to a greater impairment in executive abilities. Microbiota transplantation from human donors with increased specific Caudovirales (>90% from the Siphoviridae family) levels led to increased scores in the novel object recognition test in mice and up-regulated memory-promoting immediate early genes in the prefrontal cortex. Supplementation of the Drosophila diet with the 936 group of lactococcal Siphoviridae bacteriophages resulted in increased memory scores and upregulation of memory-involved brain genes. Thus, bacteriophages warrant consideration as novel actors in the microbiome-brain axis.