Psychobiotics and the Manipulation of Bacteria–Gut–Brain Signals

Psychobiotics are beneficial bacteria (probiotics) or support for such bacteria (prebiotics) that influence bacteria–brain relationships. Psychobiotics exert anxiolytic and antidepressant effects characterised by changes in emotional, cognitive, systemic, and neural indices. Bacteria–brain communication channels through which psychobiotics exert effects include the enteric nervous system and the immune system. Current unknowns include dose-responses and long-term effects. The definition of psychobiotics should be expanded to any exogenous influence whose effect on the brain is bacterially-mediated. Psychobiotics were previously defined as live bacteria (probiotics) which, when ingested, confer mental health benefits through interactions with commensal gut bacteria. We expand this definition to encompass prebiotics, which enhance the growth of beneficial gut bacteria. We review probiotic and prebiotic effects on emotional, cognitive, systemic, and neural variables relevant to health and disease. We discuss gut–brain signalling mechanisms enabling psychobiotic effects, such as metabolite production. Overall, knowledge of how the microbiome responds to exogenous influence remains limited. We tabulate several important research questions and issues, exploration of which will generate both mechanistic insights and facilitate future psychobiotic development. We suggest the definition of psychobiotics be expanded beyond probiotics and prebiotics to include other means of influencing the microbiome. Psychobiotics were previously defined as live bacteria (probiotics) which, when ingested, confer mental health benefits through interactions with commensal gut bacteria. We expand this definition to encompass prebiotics, which enhance the growth of beneficial gut bacteria. We review probiotic and prebiotic effects on emotional, cognitive, systemic, and neural variables relevant to health and disease. We discuss gut–brain signalling mechanisms enabling psychobiotic effects, such as metabolite production. Overall, knowledge of how the microbiome responds to exogenous influence remains limited. We tabulate several important research questions and issues, exploration of which will generate both mechanistic insights and facilitate future psychobiotic development. We suggest the definition of psychobiotics be expanded beyond probiotics and prebiotics to include other means of influencing the microbiome. The gut microbiome comprises all microorganisms and their genomes inhabiting the intestinal tract. It is a key node in the bidirectional gut–brain axis (see Glossary) that develops through early colonisation and through which the brain and gut jointly maintain an organism's health. A pivotal study found that mice raised in sterile environments and therefore lacking indigenous bacteria (germ-free mice) showed exaggerated physiological reactions to stress compared to normal controls. The abnormal reactions were reversible through probiotic-induced bacterial recolonisation [1Sudo N. et al.Postnatal microbial colonization programs the hypothalamic–pituitary–adrenal system for stress response in mice.J. Physiol. 2004; 558: 263-275Crossref PubMed Scopus (0) Google Scholar]. This finding revealed the microbiome's causal involvement in the development of the hypothalamic–pituitary–adrenal (HPA) axis. Gut bacteria have since been found to participate in the regulation of varied and important physiological processes, including immunomodulation, adiposity, and energy balance [2Hooper L.V. et al.Interactions between the microbiota and the immune system.Science. 2012; 336: 1268-1273Crossref PubMed Scopus (1137) Google Scholar, 3Kau A.L. et al.Human nutrition, the gut microbiome and the immune system.Nature. 2011; 474: 327-336Crossref PubMed Scopus (864) Google Scholar, 4Le Chatelier E. et al.Richness of human gut microbiome correlates with metabolic markers.Nature. 2013; 500: 541-546Crossref PubMed Scopus (850) Google Scholar, 5Turnbaugh P.J. et al.An obesity-associated gut microbiome with increased capacity for energy harvest.Nature. 2006; 444: 1027-1031Crossref PubMed Scopus (3862) Google Scholar] as well as the electrophysiological activity of the enteric nervous system [6Bravo J.A. et al.Communication between gastrointestinal bacteria and the nervous system.Curr. Opin. Pharmacol. 2012; 12: 667-672Crossref PubMed Scopus (0) Google Scholar, 7Foster J.A. Neufeld K.A.M. Gut–brain axis: how the microbiome influences anxiety and depression.Trends Neurosci. 2013; 36: 305-312Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar]. Probiotics, beneficial bacteria that yield positive health outcomes, have received particular attention, both in the popular press and from the research community. Here, we critically evaluate efforts to manipulate commensal gut bacteria with psychobiotics. These psychobiotics were first defined as probiotics that, when ingested in appropriate quantities, yield positive psychiatric effects in psychopathology [8Dinan T.G. et al.Psychobiotics: a novel class of psychotropic.Biol. Psychiatry. 2013; 74: 720-726Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar]. The bacteria most frequently exploited as probiotics are the Gram-positive Bifidobacterium and Lactobacillus families [9Mayer E.A. et al.Gut microbes and the brain: paradigm shift in neuroscience.J. Neurosci. 2014; 34: 15490-15496Crossref PubMed Scopus (160) Google Scholar, 10Burnet P.W. Cowen P.J. Psychobiotics highlight the pathways to happiness.Biol. Psychiatry. 2013; 74: 708-709Abstract Full Text Full Text PDF PubMed Google Scholar]. Bifidobacteria and Lactobacilli do not possess pro-inflammatory lipopolysaccharide chains, and so their propagation in the gut does not trigger full-fledged immunological reactions. With the presence of such bacteria, the immune system learns to distinguish to between pro- and anti-inflammatory entities and develops appropriate immunogenic responses by identifying pro-inflammatory elements as antigenic [11Sansonetti P.J. Medzhitov R. Learning tolerance while fighting ignorance.Cell. 2009; 138: 416-420Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar]. It should be noted, however, that Gram-positive bacteria are not always beneficial, and some, such as the Clostridia family, may be pathogenic. We propose that the definition of psychobiotics be expanded along two dimensions: First, research on healthy individuals is demonstrating that psychobiotic benefits need not be restricted to clinical groups. Second, we include prebiotics in the definition of psychobiotics. Prebiotics are compounds that, when fermented in the gut, produce specific changes in bacterial composition or activity [12Gibson G.R. et al.Dietary prebiotics: current status and new definition.Food Sci. Technol. Bull. Funct. Foods. 2010; 7: 1-19Crossref Google Scholar]. Prebiotics support the growth of intrinsic commensal bacteria. The majority of prebiotic compounds examined for their neural effects are fructans and oligosaccharides (comprising three to nine saccharide units). This review will: (i) discuss psychobiotic effects on emotional, cognitive, systemic, and central processes in animals and humans, in clinical and healthy populations, and (ii) assess the microbiome–brain signalling mechanisms enabling these effects. Much psychobiotic research is based on rodent models, which use rodent stress inductions and rodent behavioural tests to assess motivation, anxiety, and depression. Psychobiotics applied to rodent models of illness, infection, and neurodegeneration also provide early clinical insight into human diseases (Table 1). Human investigations represent a very recent trend. The psychophysiological effects of psychobiotics fall into the following three categories: (i) Psychological effects on emotional and cognitive processes. (ii) Systemic effects on the HPA axis and the glucocorticoid stress response, and inflammation which is often characterised by aberrant cytokine concentrations. Pro-inflammatory cytokines share a strong and well-studied positive association with psychiatric conditions such as depression [13Dowlati Y. et al.A meta-analysis of cytokines in major depression.Biol. Psychiatry. 2010; 67: 446-457Abstract Full Text Full Text PDF PubMed Scopus (1461) Google Scholar]. For example, injection of interferon-α, a pro-inflammatory cytokine, has been shown to induce depression, which can be alleviated through antidepressant action [14Udina M. et al.Interferon-induced depression in chronic hepatitis C: a systematic review and meta-analysis.J. Clin. Psychiatry. 2012; 73: 1128-1138Crossref PubMed Scopus (119) Google Scholar, 15McNutt M.D. et al.Neurobehavioral effects of interferon-α in patients with hepatitis C: symptom dimensions and responsiveness to paroxetine.Neuropsychopharmacology. 2012; 37: 1444-1454Crossref PubMed Scopus (0) Google Scholar]. (iii) Neural effects on neurotransmitters and proteins. Relevant neurotransmitters include γ-aminobutyric acid (GABA) and glutamate, which control neural excitation–inhibition balance. Proteins include brain-derived neurotrophic factor (BDNF), which plays a crucial role in learning and memory processes, including spatial learning, extinction of conditioned fear, and object recognition [16Lu Y. et al.BDNF: a key regulator for protein synthesis-dependent LTP and long-term memory?.Neurobiol. Learn. Mem. 2008; 89: 312-323Crossref PubMed Scopus (393) Google Scholar, 17Heldt S.A. et al.Hippocampus-specific deletion of BDNF in adult mice impairs spatial memory and extinction of aversive memories.Mol. Psychiatry. 2007; 12: 656-670Crossref PubMed Scopus (349) Google Scholar]. BDNF is reduced in anxiety and depression, a reduction that is reversible through antidepressant action [18Martinowich K. Lu B. Interaction between BDNF and serotonin: role in mood disorders.Neuropsychopharmacology. 2008; 33: 73-83Crossref PubMed Scopus (307) Google Scholar].Table 1Psychobiotics in Rodent Models of DysfunctionModelInductionPsychobioticSpeciesEffects relative to comparison groupsRefsAlzheimer's diseaseA β1–42-induced neurotoxicityPrebiotic, chitosan oligosaccharideMale Sprague–Dawley rats (n = 12)↑ Cognitive function (Morris water maze), ↓ pro-inflammatory cytokines (tumour necrosis factor-α, interleukin-1β)135Jia S. et al.Chitosan oligosaccharides alleviate cognitive deficits in an amyloid-β 1-42-induced rat model of Alzheimer disease.Int. J. Biol. Macromol. 2016; 83: 416-425Crossref PubMed Google ScholarAmyotrophic lateral sclerosisHigh level of mutant human SOD1G93A genePrebiotic, galacto-oligosaccharidesMale transgenic ALZ mice (n = 12)↓ Motor neuron death, ↓ muscular atrophy, ↑ serum folate, ↑ vitamin B12, ↑ homocysteine136Song L. et al.Galactooligosaccharide improves the animal survival and alleviates motor neuron death in SOD1 G93A mouse model of amyotrophic lateral sclerosis.Neuroscience. 2013; 246: 281-290Crossref PubMed Scopus (0) Google ScholarAutism spectrum disorderMaternal immune activationProbiotic, Bacteroides fragilisOffspring of pregnant C57BL/6N mice (n = 9–75/group)↑ Intestinal permeability, ↓ pro-inflammatory cytokines (interleukin-6), ↓ anxiety (open field test), ↓ repetitive behaviour (marble burying), ↑ communication (calling), ↑ sensorimotor gating (startle inhibition)137Hsiao E.Y. et al.Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders.Cell. 2013; 155: 1451-1463Abstract Full Text Full Text PDF PubMed Scopus (686) Google ScholarBacterial infectionCitrobacter rodentium–induced colitisProbiotic Lactobacillus rhamnosus R0011 + Lactobacillus helveticus R0052Female C57BL/6 mice (n = 4–16/group)↑ Gut barrier function, ↓ transcription of pro-inflammatory cytokines (tumour necrosis factor-α and interferon-γ, interleukin-17), ↑ transcription of anti-inflammatory cytokines (interleukin-10), normalisation of microbiome138Rodrigues D.M. et al.Probiotics are effective for the prevention and treatment of Citrobacter rodentium–induced colitis in mice.J. Infect. Dis. 2012; 206: 99-109Crossref PubMed Scopus (0) Google ScholarBacterial infectionCitrobacter rodentium–induced colitisProbiotic Lactobacillus rhamnosus R0011 + Lactobacillus helveticus R0052Neonatal C57BL/6 mice (n = 4–27/group)↓ Infection-induced death, ↓ infection-induced weight loss139Gareau M.G. et al.Probiotics prevent death caused by Citrobacter rodentium infection in neonatal mice.J. Infect. Dis. 2010; 201: 81-91Crossref PubMed Scopus (0) Google ScholarBacterial infectionCitrobacter rodentium–induced colitisProbiotic Lactobacillus reuteriMale CD1 mice (n = 105, experiment 1; n = 66, experiment 2)↓ Stress-induced gut-to-spleen pathogen migration140Mackos A.R. et al.Probiotic Lactobacillus reuteri attenuates the stressor-enhanced severity of Citrobacter rodentium infection.Infect. Immun. 2013; 81: 3253-3263Crossref PubMed Scopus (0) Google ScholarBacterial infectionCitrobacter rodentium–induced colitisProbiotic Lactobacillus reuteriMale C57BL/6 mice (n = 9/group)↓ Stress-induced infectious colitis141Mackos A.R. et al.Social stress-enhanced severity of Citrobacter rodentium-induced colitis is CCL2-dependent and attenuated by probiotic Lactobacillus reuteri.Mucosal. Immunol. 2015; 9: 515-526Crossref PubMed Google ScholarDiabetesStreptozotocin injectionProbiotics, Lactobacillus acidophilus + Bifidobacterium lactis + Lactobacillus fermentumMale Wistar rats (n = 10/group)↑ Cognitive function (Morris water maze), ↑ hippocampal long-term potentiation (LTP)142Davari S. et al.Probiotics treatment improves diabetes-induced impairment of synaptic activity and cognitive function: behavioral and electrophysiological proofs for microbiome–gut–brain axis.Neuroscience. 2013; 240: 287-296Crossref PubMed Scopus (0) Google ScholarDiabetesStreptozotocin injectionProbiotics, Lactobacillus brevis DPC 6108Male Sprague–Dawley rats (n = 10–15/group)↓ Glucose, ↓ hyperglycaemia143Marques T.M. et al.Influence of GABA and GABA-producing Lactobacillus brevis DPC 6108 on the development of diabetes in a streptozotocin rat model.Benef. Microbes. 2016; : 1-12PubMed Google ScholarHyperammonemiaAmmonium acetate injectionProbiotic, Lactobacillus helveticus NS8Male Sprague–Dawley rats (n = 6/group)↓ Inflammation (brain-inducible nitric oxide synthase, prostaglandin E2, and interleukin-1β), neurotransmitter processing (↓ abnormal metabolisation of serotonin into 5-hydroxyindole acetic acid), ↓ anxiety (elevated plus maze), ↑ cognitive function (Morris water maze)144Luo J. et al.Ingestion of Lactobacillus strain reduces anxiety and improves cognitive function in the hyperammonemia rat.Sci. China Life Sci. 2014; 57: 327-335Crossref PubMed Scopus (0) Google ScholarPost-inflammatory anxietyLipopolysaccharide injectionPrebiotic, Bimuno-galacto-oligosaccharides (B-GOS)Male CD1 mice (n = 15/group)↓ Pro-inflammatory cytokines (interleukin-1β), ↓ cortical 5-HT2A receptors145Savignac H.M. et al.Prebiotic administration normalizes lipopolysaccharide (LPS)-induced anxiety and cortical 5-HT2A receptor and IL1-β levels in male mice.Brain Behav. Immun. 2016; 52: 120-131Crossref PubMed Scopus (32) Google Scholar Open table in a new tab Psychobiotics affect psychophysiological markers of anxiety and depression. One study employed a maternal-separation model to induce early-life stress in infant male Sprague–Dawley rats [19Desbonnet L. et al.Effects of the probiotic Bifidobacterium infantis in the maternal separation model of depression.Neuroscience. 2010; 170: 1179-1188Crossref PubMed Scopus (232) Google Scholar]. Rat pups (n = 33) were either undisturbed, or, if separated, administered with either a neutral vehicle substance, an antidepressant (the selective serotonin reuptake inhibitor citalopram), or the probiotic Bifidobacterium infantis. Vehicle rats showed typical psychophysiological patterns associated with maternal separation, including poorer performance on the forced swim test and increased inflammation (indexed by heightened peripheral concentrations of the pro-inflammatory cytokine interleukin-6), decreased presence of the neurohormone noradrenaline in the brain, and elevated concentrations of amygdala corticotrophin-releasing factor messenger ribonucleic acid (mRNA). In comparison, these indices were normalised in probiotic-fed rats. Moreover, these outcomes were comparable to those observed in the citalopram group, suggesting that some probiotic benefits resemble antidepressant effects. These findings were reminiscent of earlier work [20Gareau M.G. et al.Probiotic treatment of rat pups normalises corticosterone release and ameliorates colonic dysfunction induced by maternal separation.Gut. 2007; 56: 1522-1528Crossref PubMed Scopus (188) Google Scholar], where a mixture of Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0052 downregulated HPA-axis activity (indexed by normalised corticosterone secretion) and mitigated colonic dysfunction in maternally-separated Sprague–Dawley rat pups (n = 7–15/group). In the case of nonclinical populations, experiments are defined by psychobiotic supplementation in rodents that have unimpaired psychophysiological processes and microbiomes. For example, one study examined the effects of probiotics in healthy adult male BALB/c mice (n = 36), which were innately stress-sensitive and anxious but were otherwise healthy [21Bravo J.A. et al.Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 16050-16055Crossref PubMed Scopus (703) Google Scholar]. Compared to mice administered a vehicle, those fed Lactobacillus rhamnosus JB-1 expressed fewer depressive and anxious behaviours (in the forced swim and elevated plus maze tasks, respectively). These changes were accompanied by a blunted corticosterone response to stress, suggesting that the probiotic downregulated HPA-axis activity. In the brain, probiotics differentially altered expression of inhibitory GABA receptors in a region-dependent manner. For instance, in comparison to controls, the probiotic reduced expression of GABAB1b mRNA in the hippocampus and amygdala but increased its expression in prelimbic and cingulate regions. GABA is the chief inhibitory neurotransmitter in the nervous system. Overall, probiotics may modulate regional excitation–inhibition balance, and these changes may be linked with reductions in anxiety- and depression-related behaviour and associated systemic responses. In another study, healthy adult male BALB/c mice fed Mycobacterium vaccae (n = 7–10/group across five experiments) displayed reduced anxiety in a maze-learning task [22Matthews D.M. Jenks S.M. Ingestion of Mycobacterium vaccae decreases anxiety-related behavior and improves learning in mice.Behav. Processes. 2013; 96: 27-35Crossref PubMed Scopus (0) Google Scholar]. Furthermore, performance on the maze task was substantially improved in the probiotic-fed mice, which completed the maze faster and with fewer errors, benefits that persisted at 1 week, but not 3 weeks, post-treatment. These results provide preliminary but important evidence of the potential longevity of effects, an area that has received little consideration in psychobiotic research. One programme of research [23Desbonnet L. et al.The probiotic Bifidobacteria infantis: an assessment of potential antidepressant properties in the rat.J. Psychiatr. Res. 2008; 43: 164-174Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar] investigated the effect of Bifidobacteria infantis on male Sprague–Dawley rats (n = 20) in the forced swim test, stress responses, inflammation, and monoaminergic activity. While there were no behavioural changes in swim test performance, there were significant increases of tryptophan, the serotonin precursor, in the plasma. There were also decreased concentrations of 5-hydroxyindoleacetic acid, the serotonin metabolite, in the brain. This was taken as evidence of reduced serotonergic turnover. Compared to vehicle-fed rats, blood from probiotic-fed rats exhibited reduced concentrations of the pro-inflammatory cytokines tumour necrosis factor-α, interleukin-6, and interferon-γ. These reductions jointly characterise a dampened pro-inflammatory response. This approach highlights the importance of examining physiological variables in psychobiotic research, as physiological changes were noted in the absence of significant behavioural changes. Probiotic effects during stressful experiences have also been examined [24Liang S. et al.Administration of Lactobacillus helveticus NS8 improves behavioral, cognitive, and biochemical aberrations caused by chronic restraint stress.Neuroscience. 2015; 310: 561-577Crossref PubMed Scopus (44) Google Scholar]. Healthy adult male Sprague–Dawley rats were administered Lactobacillus helveticus NS8, citalopram, or no intervention, while exposed to chronic-restraint stress (n = 24 total across treatment groups, n = 8 in an undisturbed group). Relative to the control group, the probiotic-fed rats showed lower levels of post-restraint anxiety (assessed in the elevated plus maze and the open-field test), as well as enhanced post-restraint object-recognition memory. At the biochemical level, probiotic-supplemented rats displayed lower levels of corticosterone and adrenocorticotropic hormone. The probiotic group also showed increases in the anti-inflammatory cytokine interleukin-10, and in hippocampal BDNF mRNA, noradrenaline, and serotonin. Overall, probiotic and antidepressant effects were comparable. Recent work [25Janik R. et al.Magnetic resonance spectroscopy reveals oral Lactobacillus promotion of increases in brain GABA, N-acetyl aspartate and glutamate.NeuroImage. 2016; 125: 988-995Crossref PubMed Google Scholar] has also studied psychobiotic-induced changes in central neurotransmitter concentrations in vivo using magnetic resonance spectroscopy (MRS). Healthy adult male BALB/c mice (n = 28) were administered with either Lactobacillus rhamnosus JB-1 or a vehicle for 4 weeks. Probiotic-fed mice showed elevated concentrations of glutamate and glutamine, total N-acetyl aspartate + N-acetyl aspartyl glutamic acid (tNAA), and GABA. The authors interpreted tNAA changes as a marker of alterations in neural metabolism resulting from the intervention. Glutamate is the chief excitatory neurotransmitter in the central nervous system, and, to the best of our knowledge, this is the first demonstration that it is sensitive to probiotics. The implications of concurrent elevations in both glutamate and GABA for regional excitation-inhibition balance are currently unknown, but are suggestive of an overall metabolic increase. Furthermore, because GABA and glutamate have opposing effects on neural excitability, it is possible that the total psychobiotic effect may be occurring within a zero-sum framework. The researchers also conducted periodic MRS, finding differential rates of emergence for the effects. For example, NAA increased after 2 weeks of probiotics, an elevation that was sustained for the remainder of the supplementation, and which returned to baseline 4 weeks thereafter. Glutamate and glutamine levels also increased after 2 weeks, and then remained elevated for a further 6 weeks, including 4 weeks after the intervention. Finally, GABA concentrations were only elevated in the fourth week of the intervention, but not before or after. These results represent a crucial step towards determining emergence and longevity of effects. While the general consensus is that ingestion of probiotics results in transient, rather than permanent, colonisation of the gut [26Alander M. et al.Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption.Appl. Environ. Microbiol. 1999; 65: 351-354Crossref PubMed Google Scholar], these findings suggest both that psychobiotics may have some long-term effects, and that the effects have differential longevity. A much smaller number of studies has examined the psychophysiological effects of prebiotics. These include investigations of galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS), which are a source of nutrition for Bifidobacteria and Lactobacilli, and stimulate their activity and propagation in the gut. The first report of the psychobiotic properties of prebiotics examined adult male Sprague–Dawley rats (n = 24) that were administered the Bimuno formulation of GOS (B-GOS), FOS, or water, over 5 weeks [27Savignac H.M. et al.Prebiotic feeding elevates central brain derived neurotrophic factor, N-methyl-d-aspartate receptor subunits and d-serine.Neurochem. Int. 2013; 63: 756-764Crossref PubMed Scopus (0) Google Scholar]. Relative to controls, prebiotic ingestion increased hippocampal BDNF expression and BDNF mRNA expression in the dentate gyrus. Prebiotic feeding also increased N-methyl-d-aspartate receptor (NMDAR) subunits expressed in the hippocampus. These receptors play an essential role in maintaining synaptic plasticity and optimal memory function [28Li F. Tsien J.Z. Memory and the NMDA receptors.N. Engl. J. Med. 2009; 361: 302-303Crossref PubMed Scopus (121) Google Scholar]. Both B-GOS and FOS elevated NR1 subunit expression in the hippocampus, with B-GOS additionally increasing NR2A subunits in this region, and NR1 and d-serine in the frontal cortex. The more widespread B-GOS effect, relative to FOS, may reflect the former's greater Bifidogenic capacity. There is also evidence of substantial benefits conferred by the human milk oligosaccharide 2′-fucosyllactose [29Vázquez E. et al.Effects of a human milk oligosaccharide, 2′-fucosyllactose, on hippocampal long-term potentiation and learning capabilities in rodents.J. Nutr. Biochem. 2015; 26: 455-465Crossref PubMed Scopus (16) Google Scholar]. Relative to vehicle, male rodents (both Sprague–Dawley rats and C57BL/6 mice) showed enhanced associative learning and working memory, as well as higher expression of hippocampal and striatal BDNF and increased hippocampal long-term potentiation. Prebiotic supplementation has also been studied in neonatal rats [30Williams S. et al.Neonatal prebiotic (BGOS) supplementation increases the levels of synaptophysin, GluN2A-subunits and BDNF proteins in the adult rat hippocampus.Synapse. 2016; 70: 121-124Crossref PubMed Google Scholar]. Male and female Sprague–Dawley rat pups (n = 48) were fed daily with B-GOS or a control solution from post-natal day 3 to 21. Animals supplemented with B-GOS expressed higher levels of hippocampal BDNF and NMDAR subunit GluN2A. Crucially, these changes were observed even 26 days after treatment cessation. A similar effect of human milk oligosaccharide was observed in male Lister Hooded rat pups (n = 60) [31Oliveros E. et al.Oral supplementation of 2′-fucosyllactose during lactation improves memory and learning in rats.J. Nutr. Biochem. 2016; 31: 20-27Crossref PubMed Google Scholar]. Relative to vehicle, rats fed the prebiotic during lactation showed substantially enhanced maze-learning and object-recognition one year later. These findings have important implications for assessing the longevity of prebiotic effects, and are suggestive of very long lasting gains. The rodent-human translation has been surprisingly robust, though many more human studies are necessary. In an important early investigation [32Benton D. et al.Impact of consuming a milk drink containing a probiotic on mood and cognition.Eur. J. Clin. Nutr. 2007; 61: 355-361Crossref PubMed Scopus (137) Google Scholar], male and female participants (n = 124) consumed either a fermented milk drink containing Lactobacillus casei Shirota or a placebo. At the end of the 3-week intervention, there were no overall changes in self-reported affect. However, when only participants whose baseline mood scores fell in the lowest third of the total range were analysed, probiotic supplementation resulted in significantly more participants self-rating as happy rather than depressed, relative to placebo. These results suggest that the emotional benefits of psychobiotics may be subject to ceiling effects. The researchers also found that the probiotic-fed participants performed lower on two assessments of memory function. This may be attributable to chance, as the authors themselves have suggested, but it may also imply possible detrimental effects of psychobiotics. Another well-known study provided evidence of improved mood in a generally healthy sample [33Messaoudi M. et al.Assessment of psychotropic-like properties of a probiotic formulation (Lactobacillus helveticus R0052 and Bifidobacterium longum R0175) in rats and human subjects.Br. J. Nutr. 2011; 105: 755-764Crossref PubMed Scopus (313) Google Scholar]. In a randomised and double-blind design, healthy male and female volunteers (n = 55) consumed either a mixture of probiotics (Lactobacillus helveticus R0052 and Bifidobacterium longum) or a placebo over 30 days, after which participants completed a range of self-report measures on mood and distress. Participants also collected urine over 24 hours before and after the intervention, enabling cortisol estimations. Relative to placebo, probiotic-treated participants showed significant declines in self-reported negative mood and distress. Parallel to these changes was a decrease in urinary free cortisol, which is suggestive of reduced stress. Interestingly, a follow-up analysis of the individuals with the lowest stress (indexed by cortisol concentrations) showed similar affective benefits to those with higher cortisol concentrations [34Messaoudi M. et al.Beneficial psychological effects of a probiotic formulation (Lactobacillus helveticus R0052 and Bifidobacterium longum R0175) in healthy human volunteers.Gut Microbes. 2011; 2: 256-261Crossref PubMed Google Scholar], to some extent contr