One Giant Leap from Mouse to Man: The Microbiota–Gut–Brain Axis in Mood Disorders and Translational Challenges Moving towards Human Clinical Trials

The autonomic nervous system (ANS) regulates the unconscious control of physiological homeostasis. The ENS is a network of around 500 million neurons at the interface of the gut microbiota and the host, lining the entire intestinal tract from the esophagus to the anus and which is part of the ANS. The ENS responds to receptor input from the intestine and via ganglia within the spinal cord and the brain’s medulla to coordinate various intestinal functions, such as smooth muscle activity, glandular secretion, and sphincter control [19]. Although intestinal functionality is regulated to maintain homeostasis, adaptation is possible for environmental conditions such as stress [19]. The ENS connects to the CNS (including the brain) through the vagus nerve [20], thus allowing the brain to sense the environment within the gut.

The vagus nerve is the most direct route of communication between the gut and the brain, thus enabling bidirectional communication. The vagus nerve has also been indicated in the etiology of Parkinson’s disease [21], Alzheimer’s disease [22], and depression [23]. What these conditions have in common is that they are influenced by the gut microbiota through their metabolites and immune-modulating activity. The vagus nerve functions both as a signaling pathway and transfers metabolites and other components to the brain [21]. The vagus nerve responds to components produced or induced by the gut microbiota, such as short chain fatty acids (SCFA), endotoxins, peptides, and cytokines [20]. Furthermore, neurotransmitters such as serotonin produced within the gut influence vagal functionality [23].

The gut microbiota are essential for the healthy development and function of the peripheral immune system and for the development and maturation of the innate immune cells of the brain (reviewed in [24]). Within the intestine, the mucosa provides the barrier between the inside ‘self’ and outside ‘non-self’, consisting of digesta and resident and in-coming microbes. Inflammatory responses and impairment of intestinal barrier function often go hand in hand. The induction of proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-18, and tumor necrosis factor (TNF)-α, have all been associated with depression [20,25] and increased peripheral inflammation has been observed in many psychiatric diseases such as depression, anxiety, and even autism spectrum disorder (reviewed in [26]). Circulating cytokines can access the brain through direct transportation across the blood–brain barrier (BBB). Interestingly, increased BBB permeability is a feature of many neuropathological conditions (reviewed in [26]). Modulating the microbiota may therefore reduce an inflammatory response, improve intestinal barrier function, and prevent proinflammatory cytokines directly accessing the brain. Known pathogens such as Helicobacter pylori, Clostridium perfringens, Shigella flexneri, enterohemorrhagic Escherichia coli, and enteropathogenic E. coli degrade intestinal barrier function, while organisms like Faecalibacterium prausnitzii and Akkermansia muciniphila may improve it [27]. Further, selected probiotic strains from the genera Lactobacillus (sensu lato) and Bifidobacterium have been observed to improve barrier function and exert anti-inflammatory effects [28,29].

The complex interaction between digesta and the small and large intestine induces the release of an array of gastrointestinal hormones from specialized enteroendocrine cells (EECs) distributed in the gut epithelium. These hormones, among others, regulate gastric emptying, intestinal motility, appetite, and postprandial glucose metabolism [30]. Gut microbes can influence appetite and feeding behaviors by modulating the production of such hormones from EECs.

L-cells, which are embedded mainly in the ileal and colonic epithelium, secrete glucagon-like peptide-1 (GLP-1) in response to nutrients in the small intestine. However, more distally in the intestine they are activated by luminal factors including SCFAs, bile acids, and microbial metabolic products. GLP-1 can interact with the HPA axis and the immune system [31]. In humans, GLP-1 and its receptors have been suggested to reduce anxiety [32]. Glucose-dependent insulinotropic polypeptide (GIP) is released in response to macronutrients from the enteroendocrine K-cells, distributed predominantly in the upper small intestine. GIP has been shown in animal models to reduce anxiety-like behavior [33]. Cholecystokinin (CCK) is secreted in response to the ingestion of macronutrients by enteroendocrine I-cells, located in the duodenum and upper jejunum, and stimulates the release of digestive enzymes and bile. CCK further contributes to reduced appetite [30], and has been observed to increase anxiety-like behavior [32]. Peptide YY (PYY) is co-released with GLP-1 from L-cells. PYY participates in the regulation of appetite and energy intake and has been reported to reduce stress and anxiety responses, and to improve mood [34]. Ghrelin is mainly produced in the gastric mucosa and is involved in the regulation of intestinal motility and appetite [35]. Ghrelin is found in plasma in two major forms: acyl-ghrelin increases appetite and decreases insulin secretion and sensitivity, while des-acyl-ghrelin suppresses appetite and increases insulin secretion and sensitivity [36]. Ghrelin secretion is increased in response to stress; however, chronic stress over time leads to ghrelin resistance and increased secretion of ghrelin, and this has been associated with cravings [37].

Neurotransmission, i.e., the process driving the transfer of information between neurons and their targets, can be influenced by the gut microbiota, which have been shown to produce a range of major neurotransmitters such as dopamine, norepinephrine, serotonin, γ-aminobutyric acid (GABA), nitric oxide (NO), melatonin, histamine, and acetylcholine (Ach). These neurotransmitters provide a possible mechanism of action for how the effects of the gut microbiota on mental and brain health are mediated. The most common neurotransmitters and their function are listed in Table 1.

Stress occurs when the normal homeostasis of an organism is disrupted because of an actual or perceived threat and can be categorized as either acute or chronic. Acute stress activates the HPA axis, causing an immediate release in cortisol to respond appropriately to the stressor, which can induce anti-inflammatory responses, thereby preparing the individual for defense against the presented threat. Over time, chronic stress leads to dysregulation of the HPA axis, increasing the risk of consequent side effects, such as mood and stress-related disorders, cancer [38], cardiorespiratory, metabolic, and immune system problems (reviewed in [39]). Today, chronic stress is a rapidly growing global societal challenge [40].

Stress can alter the gut–brain axis and has been shown to have a direct impact on the gut microbiota across numerous different animal models, including rodents [41,42,43] and non-human primates [44,45] (also reviewed in [46,47]). Cortisol, the primary stress hormone, has a direct influence on the ENS and vagus nerve, resulting in alterations in the gut microbiota composition [24]. In this field of research, a pioneering preclinical study conducted in germ-free (GF) mice found an exaggerated HPA axis response to stress, which could be normalized by subsequent colonization with B. infantis [48]. Acute stress has been shown to influence the microbiota community profile in mice by causing alterations in the relative proportions of the main microbiota phyla [49]. Chronic stress is linked to decreased fecal lactobacilli in rhesus macaques experiencing maternal separation early in life, concomitant with an increase in offspring stress-related behaviors [44]. Furthermore, the transfer of maternal vaginal microbiota from stressed dams to non-stressed pups resulted in an alternation in their response to stress later in life [50]. In humans, infants of mothers with high cumulative stress levels during pregnancy had an altered gut microbiota composition with lower levels of lactobacilli and bifidobacteria, higher levels of potentially pathogenic bacterial taxa, and an increase in maternally reported adverse health symptoms [51].

There is growing evidence to suggest that manipulating the gut microbiota through probiotics could modulate stress-related behavior and HPA axis activity [46]. To date, the focus has been on bifidobacteria and lactobacilli, with both preclinical and clinical studies demonstrating promising effects on stress and psychiatric disorders such as anxiety and depression [24,47,52]. In this regard, preclinical models have shown beneficial effects of bifidobacteria and lactobacilli to ameliorate stress-induced behavioral alterations across the lifespan, indicative of a link between the gut microbiota and the stress response. For example, Companilactobacillus farciminis prevented the hyperactivation of the HPA axis elicited by acute stress, which the authors hypothesized was a result of the prevention of excessive gut permeability associated with acute stress [53]. Sprague Dawley rats exposed to chronic restraint stress also showed improved anxiety- and depression-like behavior and improved cognitive function following administration of Lactobacillus helveticus MCC1848 [54]. Lactiplantibacillus plantarum supplementation alleviated heightened stress responses as a result of both chronic unpredictable stress and sleep deprivation stress [55]. Supplementation with Bifidobacterium spp. has also been reported to alleviate stress-induced behavioral alterations in preclinical models [56,57].

Moving from preclinical to clinical evidence, probiotics have been proven to have some success in ameliorating mood in a number of clinical studies [58,59]. Improvements in mood scores were observed in elderly participants following administration with a milk drink containing Lacticaseibacillus casei, proving most beneficial in the participants that reported the lowest mood scores at baseline [60]. A multi-species combination of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis, Lactobacillus acidophilus, L. plantarum, Bifidobacterium animalis subsp. lactis and Limosilactobacillus reuteri administered to healthy participants elicited anxiolytic effects [61], whereas another multi-species combination (nine strains, including Lactobacillus (sensu lato), Lactococcus, and Bifidobacterium) ameliorated cognitive reactivity to sad mood in healthy participants [62]. Of note, one later study using the same combination demonstrated that the neurocognitive benefits of this multi-species probiotic became evident only when the participants were stressed, highlighting the need to carefully characterize study populations [63]. A multi-species combination (containing L. fermentum LF16, Lacticaseibacillus rhamnosus LR06, L. plantarum LP01, and B. longum BL04) induced significant improvements in mood, with a reduction in depressive mood state, anger, and fatigue, and an improvement in sleep quality in healthy volunteers [64]. L. plantarum DR7 administration to stressed adults alleviated stress and anxiety, as well as improving several aspects of memory and cognition, enhanced serotonergic signaling, and decreased plasma cortisol and proinflammatory cytokines [65]. Intake of L. plantarum HEAL9 also led to a significant decrease in the plasma levels of two inflammatory markers (soluble fractalkine and CD163) following exposure to an acute stress test [66]. A 12-week intervention with Lactobacillus gasseri and B. longum also resulted in positive changes in stress and salivary cortisol measurements and concomitant improvements in immune response in healthy participants [67]. Healthy medical students undergoing university examinations had reduced levels of stress following the consumption of a fermented milk containing the probiotic L. casei Shirota [68]. Furthermore, increases in salivary cortisol reported during an exam stress period were also reduced in a healthy student population supplemented with L. plantarum 299v [69]. A multi-species probiotic administered to healthy college students was found to improve panic anxiety, neurophysiological anxiety, negative affect, worry, and increase negative mood regulation [70]. Further, supplementation with L. casei [71] and Bifidobacterium bifidum [72] reduced the physical symptoms of exam stress, including the onset of stress-induced gastrointestinal symptoms and head colds. Finally, an open-label study conducted in highly stressed information technology specialists found that administration of L. plantarum PS128 improved several self-reported and objective measures of mood, anxiety, stress, and sleep [73].

Taken together, these examples highlight significant results and describe an intriguing role of probiotics in mood, anxiety, stress, and related behaviors such as sleep. A recent meta-analysis demonstrated that probiotic consumption could result in a reduction of subjective stress levels in healthy volunteers and may alleviate stress-related subthreshold anxiety and depression levels [58]. However, further clinical studies are required to provide a deeper understanding of the strain specificity and mechanisms of action of probiotics to help fully realize their role in stress management and the relief of the symptoms of anxiety.

Major Depressive Disorder (MDD) is a common psychiatric disorder characterized by depressed mood or significantly reduced pleasure or interest in all activities and is currently a leading cause of disability worldwide. Emerging evidence shows that the dysfunction of the gut–brain axis may be implicated in the etiology of depression. In support of this, the gut microbiota are impacted by MDD and associated with changes to gut epithelial permeability and increased systemic inflammation with elevated levels of C-reactive protein, IL-1β, IL-6, and TNFα in depressed patients compared with healthy controls [74]. Furthermore, the “leaky gut” phenomenon resulting from disrupted gut barrier function is proposed to contribute to MDD. In this context, MDD patients show elevated serum concentrations of immunoglobulin (Ig)-M and IgA against lipopolysaccharides of Gram-negative bacteria compared to healthy controls [75], suggesting an increase in bacterial translocation from the gut and subsequent inflammatory response, potentially contributing to an MDD phenotype.

Preclinical models of depression, such as the maternal separation model [76] and the Flinders-sensitive rat model [77], have demonstrated alterations in the gut microbiota composition and inflammation. In humans, many studies have examined alterations in the gut microbiota in MDD patients compared to healthy controls [78,79,80,81]. Specifically, MDD patients are reported to have an altered gut microbial compositional profile relative to healthy controls [80,81,82]. Patients with MDD have been reported to have reduced abundances of Bacteroidetes, Firmicutes, and Actinobacteria with a concomitant outgrowth of Proteobacteria [81,82], and increased abundance levels of Alistipes spp. [80]. The Flemish Gut Flora project provided further associations between the gut microbiota profile in a depressive cohort by highlighting the absence of Coprococcus and Dialister species in patients with depression [83]. However, several variations have been reported across these studies, which may be due to the small sample sizes or the effects of adjunct medications [47].

The use of probiotics for the reduction of symptom severity in MDD has gained attention in recent years, indicated by increasing numbers of preclinical and clinical studies that have supported the anti-depressive efficacy of probiotics. Accumulating preclinical evidence indicates that single-strain or multi-species preparations may be effective in improving the behaviors related to depression (reviewed in [47]). In a recent study, a multi-species probiotic combination of L. plantarum LP3, L. rhamnosus LR5, B. lactis BL3, B. breve BR3 and Pediococcus pentosaceus PP1 alleviated depressive-like behaviors and decreased corticosterone levels in mice subjected to restraint stress [84]. Similarly, L. plantarum WLPL04 alleviated anxiety- and depressive-like behaviors and chronic stress-induced cognitive dysfunction in mice, while also reversing abnormal alterations in the composition of the gut microbiota [85].

In humans, administration of B. longum NCC3001 for six weeks to adults with irritable bowel syndrome (IBS) and mild to moderate anxiety and/or depression reduced depression scores and enhanced the participants’ quality of life, which was associated with alterations in brain activation patterns in the limbic system. No improvement in anxiety scores were observed in this cohort [86]. Another study investigated the effect of B. coagulans MTCC 5856 in patients experiencing co-morbid IBS symptoms with MDD and found that the probiotic significantly improved symptoms of both depression and IBS [87]. Slykerman and colleagues found that L. rhamnosus HN001 supplementation during pregnancy resulted in a significant reduction of postnatal depression and anxiety symptoms [88]. An improvement in cognition was reported in a cohort of depressed patients receiving L. plantarum 299v compared to the placebo group [89]. MDD patients who were administered L. acidophilus, L. casei, and B. bifidum for eight weeks also reported ameliorations in self-reported depression scores [90]. Finally, an open-label study conducted in patients with treatment-resistant depression highlighted the potential of probiotics as an adjunct therapy with antidepressant drugs [91].

Further evidence to support these clinical findings are reported in several systemic reviews [92,93,94]. However, it is important to note that several intervention studies failed to demonstrate any beneficial effects on improving overall mood (reviewed in [95,96,97]). It is evident from the preclinical research that specific bacterial strains play a role in ameliorating depressive-like behaviors, and certain clinical studies have demonstrated a role for probiotics towards alleviating symptoms of depression; however, the exact bacterial species and/or strains and mechanisms underpinning their beneficial effects remain unclear. Nevertheless, current research highlights the importance of a healthy microbiome for patients suffering from depression. Future studies into the strain-dependent nature of putative probiotics in patients with clinically diagnosed MDD are warranted to evaluate their therapeutic potential.

GF animals are those lacking any microbial exposure since birth, and although extremely abnormal, research in GF mice has significantly enhanced our understanding of the crucial role of microorganisms across virtually all physiological processes in the host [98]. GF mice have revealed the importance of the microbiota for normal aging, immune-, metabolic-, digestive-, and gastrointestinal function, and the normal development and function of the nervous system [99]. For an extensive overview of the impact of the complete absence of gut microbiota on brain physiology, function, and behavior of the GF mouse, please see [98].

Sudo and colleagues were the first to demonstrate that the exposure of GF mice to an acute experimental stressor resulted in an exaggerated increase in glucocorticoid production from the HPA axis compared with SPF control mice [48]. Interestingly, monocolonization with either B. infantis or enteropathogenic E. coli in early life could either normalize or further exacerbate the HPA axis response to stress observed in GF mice, respectively [48]. This effect of the GF condition on stress responsivity has since been reproduced in a different mouse strains [100] and in rats [101].

Research in GF models has enhanced our knowledge of the microbiota–gut–brain axis and has provided the most convincing evidence towards elucidating the pathways of microbial impact on the development and function of several key physiological systems, in this case the impact on the nervous system. These important discoveries in GF models can be used to guide research and innovation aimed at developing new therapeutic solutions that target the gut microbiota and impact brain health, but since the GF condition has such far reaching consequences on the body as a system, the translatability to the clinical condition is very limited.

Unquestionably, antibiotics are one of the most important influencing factors on the gut microbiome, and in addition to GF models, antibiotic models have provided us with another useful tool for investigating the impact of the gut microbiome on health. Antibiotic models enable more specific targeted disruption of the gut microbiota as opposed to the complete absence of gut microbiota in the GF model. In the context of translation, antibiotic models can be tailored to resemble the clinical scenario in humans more closely. For example, the extent to which the microbiota are depleted, the dose of antibiotics used, the duration of treatment, the composition of antibiotic used (single or cocktail), and the timing of life during which the antibiotics are administered can all be controlled to mimic various clinical scenarios [47]. Such methodological tweaks enable researchers to determine the effect of antibiotic-associated gut microbiome disruptions on brain functions and behavior.

The collective studies using antibiotic models (Table 2) provide significant evidence that gut microbiota depletion during critical windows of development—potentially already starting at the periconception period, but certainly into adolescence—significantly impacts brain function and behavior throughout life. Antibiotic administration also has an impact on immune, behavioral, and neurochemical markers in the brain [102,103,104,105,106,107]. Irrespective of the methods used for microbiota depletion, these findings have potentially significant translational relevance considering the clinical use of antibiotics in both mothers and infants. Although these results are intriguing, and certainly describe a role of the gut microbiome in neurodevelopment in early life, symptom onset, and the progression of brain disorders, it must be acknowledged that the clinical setting in all cases is much more complex and thus, the translation of these findings is complicated.

In the context of the microbiota–gut–brain axis and mechanistic studies to underpin the role of the gut microbiome in brain health, animals, most commonly mice, can be humanized following engraftment with parts of the human gut microbiota. This is done through fecal microbiota transplant (FMT), the process of transferring the gut microbiota from humans into either a GF mouse or a mouse pretreated with antibiotics to deplete the murine gut microbiota. The subsequent repopulation of the mouse gastrointestinal tract with the microorganisms transplanted from the human fecal matter results in the generation of a humanized mouse with a gut microbiota resembling that of the human donor [9]. The translational challenge is, of course, that the gut microbiome is the only humanized component of the rodent, and the background of the recipient mouse should also be seriously considered. Prior to the FMT, GF recipient mice are already markedly altered and depending on the dose, duration, and composition of the antibiotic cocktail, antibiotic-treated recipient mice may have experienced other CNS effects or incomplete microbiome depletion. Settanni et al. recently reviewed the evidence on FMT for brain health and extensively documented the studies in which FMT has been used to induce psychiatric symptoms in rodent models [108]. The gut microbiota from donors with depression [81,82], alcoholism [109,110], anorexia nervosa [111], IBS [112], and schizophrenia [113,114] have all been transplanted into rodents that later presented abnormalities in behavior, indicating at least partial transfer of the clinical psychiatric phenotype. Although not all of the phenotypic characteristics of patients with depressive disorder were transplanted via the microbiota, and the number of donors in both studies was low [81,82], these models do provide some opportunity to investigate microbiome-associated depression and novel microbiome-targeted therapeutics such as probiotics and custom diets. One major challenge with FMT studies is the engraftment of the donor microbiota in the host; thus, booster inoculations are often administered to curtail this [81].

Outside of humanized rodent models, FMT has been used most frequently and successfully in the clinical setting to treat recurrent Clostridiodes difficile infection [115] and has been investigated in a limited capacity to treat psychiatric disorders (extensively reviewed in [108]). In addition, FMT has also been performed within species to demonstrate the transplantation of an anxious phenotype from a mouse strain (BALB/c), that displays a more innately anxiety-like behavior, to a GF model of a mouse strain (NIH Swiss), that displays less anxiety-like behavior, thus further implicating the microbiome in anxiety [116]. FMT has also been used to demonstrate the impact of prenatal stress on the maternal microbiome, maternal-to-offspring microbiota transmission, and associated stress-related deficits on the offspring’s brain and gut [50].

Overall, humanized (gut) rodent models have led to several breakthrough discoveries and have significantly enhanced our understanding of the role of the gut microbiome in brain health. Although the translation to the clinical setting is challenging, these models should be considered within preclinical study design as a crucial step towards bridging the gap between mouse and man.

B. longum 1714^® has been investigated across several preclinical and clinical trials with successful translational results. Here, we will review the psychobiotic evidence for this strain, starting with preclinical models and then moving towards clinical populations.

Innately anxious but otherwise healthy BALB/c mice were administered B. longum 1714 at a dose of 1 × 10⁹ colony forming units (CFU) per day, or a commonly prescribed selective serotonin reuptake inhibitor and compared to a vehicle group. While B. longum 1714 and escitalopram reduced compulsive and anxiety-like behavior in the marble burying test, subsequent behavioral tests showed that the strain further reduced stress- and depression-related behavior more efficiently than the antidepressant drug [57]. In a follow-up study, improvements in several aspects of cognitive function were demonstrated in BALB/c mice that were administered B. longum 1714, at the same dose as previously described, compared to a vehicle group [150]. The authors concluded that the impact of B. longum 1714 on cognitive function could be associated with reduced anxiety. Taken together, these preclinical findings highlighted B. longum 1714 as a candidate psychobiotic worthy of clinical investigation.

In the first clinical trial, healthy volunteers were recruited for a within-participants, repeated measures, placebo-controlled clinical trial to investigate the effects of B. longum 1714 on the stress response, cognition, and brain activity patterns [151]. At baseline, the participants completed the socially evaluated cold pressor test (SECPT) coupled with the state portion of the state-trait anxiety inventory to measure the combined psychological and physiological response to an acute stress, and a resting electroencephalography (EEG) recording was taken to measure brain activity prior to cognitive assessments. The participants received a placebo for the first four weeks, followed by a four-week intervention of B. longum 1714 at a daily dose of 1 × 10⁹ CFU. Further, the study had a two-week follow-up after probiotic administration. B. longum 1714 reduced cortisol output during the SECPT compared to placebo and the baseline assessment. Subjective anxiety was significantly increased in response to the acute stress at baseline and following intervention with placebo; however, this was attenuated following intervention with B. longum 1714. Perceived stress was marginally reduced following the B. longum 1714 intervention compared to placebo, and perceived stress levels also increased in the two-week follow-up post B. longum 1714 intervention. Finally, B. longum 1714 improved visuospatial memory performance that was coupled with an EEG profile consistent with improved memory. These results indicated the early psychobiotic potential of B. longum 1714, consistent with the preclinical findings [151]. A subsequent randomized, double-blind, placebo-controlled clinical trial involved healthy adults allocated to receive either a placebo or the same dose of B. longum 1714 for four weeks [152]. Brain activity was measured using magnetoencephalography in response to social stress characterized by social exclusion and rejection induced by a standardized social stress paradigm. In addition, the participants health status was measured at baseline and at the end of the study using a self-report questionnaire. Although there was no effect overall on health status, B. longum 1714 modulated resting state neural activity, which correlated with enhanced energy/vitality [152]. Furthermore, all participants experienced an increase in social stress in response to the social stress paradigm, but B. longum 1714 altered the neural responses during social stress—brain activity which may play a role in the activation of brain-coping centers to counter-regulate negative emotions [152]. However, Moloney et al. [153] conducted a randomized, placebo-controlled, repeated measures study where healthy students consumed both placebo and B. longum 1714 for eight weeks in a crossover design during their preparation for university semester examinations. The post-intervention assessments took place during the examination period, a naturalistic chronic stressor in this study. Self-reported stress, anxiety and depression, and cortisol output only marginally increased from a very low baseline level in response to exam stress, and B. longum 1714 did not alleviate any of these symptoms [153]. A positive effect of B. longum 1714 compared to placebo on sleep duration was observed when analyzing the change from baseline scores [153]. Given the differences in the model of prolonged chronic stress in preparation for university semester examinations and the acute stress tests that were applied in the previous studies, it could be hypothesized that candidate psychobiotics could be more effective in participants with moderate anxiety or under exposure to an artificial stressor.

L. paracasei Lpc-37^® has been investigated across two preclinical studies using the same chronic stress model and in one clinical trial with healthy participants. The translation of the results from preclinical to clinical will be discussed here.

Chronically stressed mice were administered one of twelve candidate strains at a daily dose of 1 × 10⁹ CFU for a total of five weeks, after which behavior and the neuroendocrine response to stress were investigated. Of the twelve strains, L. paracasei Lpc-37, L. plantarum LP12407, and L. plantarum LP12418 were most effective at attenuating anxiety- and depression-related behavior following chronic stress, and this was observed across two independent experiments in the same model [154]. A reduction in corticosterone was observed following intervention with L. paracasei Lpc-37 [154]. Crucially, there were eight candidate probiotic strains included in this large screening experiment that had no effect on anxiety- and depression-related behavior, highlighting the challenges in discovering a candidate psychobiotic, even in a model where mice display anxiety-like behavior and where environmental factors such as diet are tightly controlled.

A randomized, double-blind, placebo-controlled clinical trial was established whereby healthy participants (n = 118) received either placebo or L. paracasei Lpc-37 at 1.75 × 10¹⁰ CFU per day for five weeks [155]. It was observed that self-reported perceived stress was significantly reduced with L. paracasei Lpc-37 compared to the placebo group [155]. L. paracasei Lpc-37 was observed to reduce the increase in heart rate in response to the acute stress in participants with low chronic stress; the opposite was observed in participants with high stress levels. Such a result could suggest that the effect of L. paracasei Lpc-37 on ANS response to stress based on heart rate may be differentially dependent on chronic stress. Further significant effects were identified within the subgroups, where it was shown that L. paracasei Lpc-37 increased perceived productivity, feelings of restfulness after a night’s sleep, and perceived health and reduced diastolic blood pressure in participants with high stress levels compared to the placebo group. In participants with low stress levels, L. paracasei Lpc-37 reduced fatigue levels during the acute stress procedure and normalized evening cortisol levels. Furthermore, L. paracasei Lpc-37 was also shown to significantly reduce perceived stress in females following the five-week intervention in comparison to the placebo group, whereby females could be considered a more stress-vulnerable population in this study [155]. Taken together, the results of these studies highlight L. paracasei Lpc-37 as an interesting psychobiotic candidate with limited, but consistent, translation of stress-reducing results from a preclinical model to a clinical trial.

The combination of L. helveticus Rosell^®-52 (R0052) and B. longum Rosell^®-175 (R0175) has been investigated across various preclinical models and clinical trials of healthy and patient populations for several years, with some translational findings, but other conflicting results.

The combination of L. helveticus R0052 and B. longum R0175 was first investigated in different experiments of myocardial infarction (MI) [156,157,158]. It was observed that administration of the probiotic combination for just seven days prior to inducing a MI, and then again for ten days beginning from the seventh day post-MI until euthanasia, prevented MI-induced depression-like behavior and deficits in social interaction from developing and restored intestinal barrier integrity in MI rats [156]. A subsequent study showed that the probiotic combination reduced pro-apoptotic activity within specific brain regions, and attenuated deficits in social interaction behavior and depression-like behavior [157]. Later, it was discovered that the beneficial effects of the probiotic combination on preventing the symptoms of MI developing were dependent on the integrity of the vagus nerve [158].

Other early investigations demonstrated that a two-week administration of the probiotic combination reduced anxiety-like behavior in rats [159]. In a mouse model of chronic psychological stress, a significant reduction in stress-induced increases in plasma corticosterone and catecholamines was noted, and modulated specific markers of neuronal activity and attenuated stress-induced alterations in hypothalamic synaptic-plasticity-related neuronal networks, stress-induced deficits in hippocampal neurogenesis, and stress-induced intestinal permeability were observed [160]. In a follow-up experiment using the same design and model, the probiotic combination was shown to attenuate chronic stress-induced visceral hypersensitivity and increases in plasma corticosterone and catecholamines. Moreover, the probiotic combination also prevented the stress-induced reduction in the mRNA expression of the glucocorticoid receptor in several brain regions associated with the stress response. [161]. In a model of LPS-induced peripheral and neuroinflammation, two weeks of pretreatment with the probiotic combination reduced the circulating and protein expression levels of pro-inflammatory cytokines in the hippocampus [162]. Although systemic exposure to LPS induced deficits to cognitive function, the probiotic combination demonstrated no effect at the behavioral level but did prevent LPS-induced deficits in BDNF expression levels in the hippocampus [162]. In a genetic rodent model of depression, the probiotic combination reduced the depression-associated increase in noradrenaline levels in the plasma and reduced the increase in dopamine levels in the plasma. Neither dose of the probiotic combination had any effect on brain monoamine levels or cognition and anxiety- or depression-like behaviors [163]. In a hamster model of social defeat stress, the probiotic combination significantly increased social avoidance and decreased social interaction in hamsters who displayed anxiety-like behavior, whereby this increase in social anxiety-like behavior was associated with reduced microbial diversity following social defeat [164]. This behavioral result somewhat conflicts with previous studies, which reported a reduction in anxiety-like behavior following intervention with the probiotic combination.

The potential psychobiotic effect of L. helveticus R0052 and B. longum R0175 in combination has also been investigated in clinical trials. Healthy adults affected by self-reported daily stress reported significantly reduced abdominal pain and nausea/vomiting compared to the placebo group [165]. There was no effect of the probiotic combination on any of the other gastrointestinal symptoms of stress that were assessed or on any of the other physical or psychological discomforts induced by stress [165]. Healthy but borderline clinically anxious and depressed adults consuming the probiotic combination reported a greater reduction in the median global symptom severity index percentage change scores following intervention compared to the placebo group. This result was attributed to greater reductions in the percentage change scores for somatization, depression, and anger–hostility. Furthermore, the median percentage change for the global Hospital Anxiety and Depression Scale (HADS) score in participants who were supplemented with the probiotic combination was reduced more than that of the placebo group from baseline to the end of the study. However, it should be noted that the median HADS global score was exactly the same within both groups at the end of the study [159]. In a post hoc analysis conducted on “less stressed” participants, the percentage change scores for perceived stress and obsessive–compulsive, anxiety, and paranoid ideation were further reduced significantly following supplementation with the probiotic combination compared to placebo [166].

Depressive patients free of any psychiatric medication were recruited for the next randomized, double-blind, placebo-controlled trial to investigate the effects of 3 × 10⁹ CFU per day of L. helveticus R0052 and B. longum R0175 for eight weeks. There was no significant effect of the probiotic combination on any of the nine psychological outcome measures included in the study, or on any blood-based biomarker (CRP, IL-1β, IL-6, TNF-α, and BDNF) [97]. However, in the probiotic group, it was observed that participants with high levels of vitamin D at baseline experienced a significantly greater improvement in several psychological outcomes over time compared to those with low baseline levels of vitamin D [97]. The probiotic combination was next investigated in patients with MDD who were taking antidepressant drugs for three months or more before the trial commenced. The combination of L. helveticus R0052 and B. longum R0175 significantly reduced depressive symptoms as measured using the Beck Depression Inventory (BDI), compared to the placebo group [167]. Lastly, the probiotic combination was investigated in treatment-naïve patients with MDD. In both studies, the effect of the probiotic combination at the dose of 3 × 10⁹ CFU per day for eight weeks was investigated in 10 patients per study with a score of ≥20 in the Montgomery Åsberg Depression Rating Scale indicating at least moderate depression. The studies showed a significant improvement in subjective psychological outcomes, but there was no effect on objective measures of sleep quality [168,169]. It is, of course, unclear to what extent these outcomes are influenced by a placebo effect.

There are already substantial data concerning the psychobiotic potential of L. helveticus R0052 and B. longum R0175. Based on the evidence discussed above, L. helveticus R0052 and B. longum R0175 have demonstrated some conflicting results in both preclinical and clinical trials, and the psychobiotic potential of this probiotic formulation is worth investigating further in future clinical trials.

L. rhamnosus JB-1™ (previously referred to as L. reuteri) has comprehensive preclinical evidence that repeatedly demonstrates behavioral, physiological, and neurobiological efficacy. Despite this, L. rhamnosus JB-1 has only been investigated in one clinical trial to date, which reported an unsuccessful translation of results.

It was first shown that L. rhamnosus JB-1 inhibited the cardio–autonomic response to colorectal distension (CRD) and the perception of visceral pain evoked by CRD [170]. This discovery led to a hypothesis that L. rhamnosus JB-1 could be used as a potential treatment for patients with functional bowel disorders, such as IBS, experiencing abdominal discomfort and pain. It was later shown that the nociceptive effects of L. rhamnosus JB-1 resulted from a reduction in CRD-induced dorsal root ganglia excitability [171], and that the strain targets an ion channel in enteric sensory nerves to influence gut motility and pain perception [172]. In addition, the luminal application of L. rhamnosus JB-1 to naïve mouse jejunum and colon tissue samples affected gut motility in ex vivo perfusion models [173,174]. In a similar ex vivo model, this time using naïve tissue samples taken from mice previously exposed to an acutely stressful procedure, it was shown that L. rhamnosus JB-1 countered the effect of stress-induced dysmotility in both jejunal and colon segments [175]. Taken together, these results point to the ENS and spinal pathways as a potential pathway for gut–brain communication by L. rhamnosus JB-1.

L. rhamnosus JB-1 reduced stress-induced corticosterone and anxiety- and depression-like behavior in BALB/c mice innately displaying anxiety-like behavior. This effect on the stress response and behavior was coupled with a reduced GABA_B1β receptor expression in the hippocampus and amygdala, which is consistent with the anti-depressant like effect of GABA_B receptor antagonists [176]. Remarkably, it was demonstrated that these neurochemical and behavioral effects were not detected in vagotomized mice, thus identifying the vagus nerve as an essential route of gut–brain communication through which L. rhamnosus JB-1 mediated effects on brain function and behavior [176,177]. In a separate experiment, magnetic resonance spectroscopy (MRS) later confirmed that intervention with the same dose of L. rhamnosus JB-1, also in BALB/c mice, increased levels of three biomarkers of brain function, including glutamate/glutamine and GABA [178]. In a model of chronic psychosocial stress, L. rhamnosus JB-1 attenuated stress-induced anxiety-like behavior and deficits in social interaction [179,180,181], coupled with immuno-regulatory action and a dampening of the stress-induced immune response [179], a normalization of serum kynurenine and kynurenic acid, and a restoration of stress-induced abnormalities in tryptophan-kynurenine metabolism [180]. This was independent of any preventative effects on stress-induced dysbiosis [179]. However, the administration of L. rhamnosus JB-1 after the social defeat stress perpetuated, rather than prevented, the behavioral and physiological deficits associated with stress exposure [182]. A similar result was demonstrated for the selective serotonin reuptake inhibitor (SSRI) sertraline, indicating that the timing of the intervention is critically important.

Early life concurrent administration of L. rhamnosus JB-1 to antibiotic-treated pregnant dams until weaning decreased anxiety-like behavior in female offspring, and prevented antibiotic-associated deficits in sociability [136]. Administering L. rhamnosus JB-1 to antibiotic-treated offspring one week prior to weaning prevented the antibiotic-associated effects, such as deficits in social behavior (but not on anxiety-like behavior), changes in gene expression in specific brain regions, and changes in immune cell populations in the spleen [183].

On a general level, when comparing the different mouse models, it appears that BALB/c mice [176] are more responsive compared to C57BL/6 [179] and Swiss Webster mice [184]. BALB/c mice are often preferentially selected as animal models of anxiety and depression, as they better reflect the core features observed in these patient populations [184]. When there are differences between mice strains, it is obvious that translation to humans is even more prone to challenges.

Healthy male participants received L. rhamnosus JB-1 for eight weeks and were exposed to an acute stressor; the influence on cognitive function, anxiety, mood, host inflammatory profile, and brain activity patterns measured using EEG were determined [185]. L. rhamnosus JB-1 was not found to have an effect on mood, anxiety, stress, sleep quality, memory performance, attention, or anti-inflammatory cytokine levels compared with the placebo. Nor was there any effect on either the psychological response or the neuroendocrine response to the acute stress [185]. In the preclinical studies, L. rhamnosus JB-1 only demonstrated beneficial effects on behavior, physiology, and brain function in either stress-sensitive mice or in models of chronic stress. This is very different compared to the healthy human participants that made up the population in this study [185].

This study highlights the fundamental challenges in translating the findings from putative psychobiotic candidates in stress-susceptible animals to healthy human populations. Nevertheless, the preclinical evidence on the psychobiotic potential of L. rhamnosus JB-1 is substantial. However, it may be advised that future clinical trials focus on more clinically anxious populations, or in patients with mood disorders such as anxiety and depression.