Brain–gut–microbiota axis in depression: A historical overview and future directions

Depression is the most common mental disorder and a leading cause of disability worldwide. Despite abundant research, the precise mechanisms underlying the pathophysiology of depression remain elusive. Accumulating evidence from preclinical and clinical studies suggests that alterations in the gut microbiota, microbe-derived short-chain fatty acids, D-amino acids and metabolites play a key role in the pathophysiology of depression via the brain-gut-microbiota axis, including the neural and immune systems. Notably, the brain-gut-microbiota axis might play a crucial role in susceptibility versus resilience in rodents exposed to stress. Vagotomy is reported to block depression-like phenotypes in rodents after fecal microbiota transplantation of "depression-related" microbiome, suggesting that the vagus nerve influences depression through the brain-gut-microbiota axis. In this article, we review recent findings regarding the brain-gut-microbiota axis in depression and discuss its potential as a therapeutic target for depression.


Introduction
Depression has received substantial attention from researchers and the general public, particularly due to its link to suicide. It is estimated that 280 million people of all ages, and 5.0% of adults, are affected by depression worldwide (Global Health Data Exchange, 2021). This high global burden of disease dramatically affects individuals' and families' abilities to live a rewarding life (GBD, 2019 Disease andInjuries Collaborators, 2020;Friedrich, 2017;Global Health Data Exchange, 2021). In 2020, 5683 unique data sources from the global burden of disease study showed that the prevalence of depression or anxiety disorders has significantly increased due to the COVID-19 pandemic (COVID-19 Mental Disorders Collaborators, 2021). At present, the available treatments operate on the main principle of abnormal brain chemistry in depression. Due to treatment resistant depression and the delayed onset of first-line antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs), the needs of many depression patients are unmet, even with complementary therapies such as inter-personal psychotherapy and non-pharmacological therapy such as electroconvulsive therapy (Beurel et al., 2020;Wei et al., 2021). One challenge is that the precise mechanisms underlying the pathophysiology of depression remain unknown. Unlocking the biological mechanisms of depression is still a work in progress. A new perspective targeting the brain-gut-microbiota axis might more closely resemble the ground truth in depression, and could thus contribute to the development of new antidepressant drugs.
In this article, we review the role of the brain-gut-microbiota axis in depression. This article provides a summary of the scientific basis that the brain-gut-microbiota axis influences mental health disorders, including depression (Lach et al., 2018;Martin et al., 2018). We then discuss the bidirectional and mutually interacting network targeting the brain-gut-microbiota axis in depression.

The brain-gut-microbiota system
In 1980, the concept of a gut-brain axis was unexpectedly born due to research findings on hormone signaling of the gastrointestinal endocrine system in neurons and brain cells (Track, 1980). In the following decades, the concept has been further strengthened and expanded to include the microbiome's contribution to the gut-brain axis (Carabotti et al., 2015;Hsiao et al., 2013;Margolis et al., 2021). At present, the brain-gut-microbiota denotes more than just the concept of the brain-gut-microbiota axis, it also represents the brain-gut-microbiota system in hosts and interactions among central nervous system (CNS), endocrine chemical signal system, immune regulation, microbiota and metabolic effects, and barrier functions in the brain and gut (Fig. 1). The coordination of these factors plays an important role in maintaining an individual's state of health (Foster and McVey Neufeld, 2013;Lynch and Pedersen, 2016;Maiuolo et al., 2021;Margolis et al., 2021). If imbalance exists in the brain-gut-microbiota axis, various kinds of diseases-including mental health disorders like depression-may occur (Foster and McVey Neufeld, 2013;Lynch and Pedersen, 2016;Maiuolo et al., 2021;Margolis et al., 2021).
The microbiome is mainly made up of bacteria, viruses, archaea, and The role of the brain-gut-microbiota axis in the body. The major pathways of the brain-gut-microbiota axis are shown among the central nervous system (CNS), periphery, and gut. The network of neural signals, immune signals, and chemical signals constitutes a major part of the brain-gut-microbiota axis. Alterations in this network involves complex interactions across organs by direct (nerves) and indirect (systemic circulation) pathways, contributing to altered homeostasis in the body. Changes in the gut microbiota composition and microbiome-derived SCFAs, D-amino acids and metabolites can alter neural signals, immune signals, and chemical signals to cause depression. The CNS works through neural regulation, neurotransmitters, immune signals, and other factors that target peripheral organs and tissue in response to different signal reflexes, thereby contributing to normal bidirectional brain-gut axis signaling. When this balance is destroyed by stress, depression or other diseases may occur. CNS: central nervous system, 5-HT: 5-hydroxytryptamine, IL-6: interleukin 6, IL-17: interleukin 17, IL-1β: interleukin 1β, ILC: innate lymphoid cells, LPS: lipopolysaccharide, SCFA: short chain fatty acid, TMAO: trimethylamine N-oxide, TNF-α: tumor necrosis factor α. Some materials of the figure have been designed by using the resources from Freepik.com.
fungi at the order of magnitude of a trillion microbes; as a result, the ratio is nearly 1:1 of bacterial to human cells in the body, although the genomic content of the microbiome contains more than 100-fold more genes than the human genome (Douglas et al., 2020;Lynch and Pedersen, 2016;Sender et al., 2016;Thursby and Juge, 2017;Zhu et al., 2010). The gut microbiota plays essential roles involving digestion and absorption from the diet, defending the gastrointestinal tract from pathogens, and secreting essential vitamins such as folic acid and vitamin K for overall bodily health (O'Hara and Shanahan, 2006;Rowland et al., 2018). In recent years, more and more studies have found that the gut microbiota and host have a mutually beneficial relationship starting from the earliest stages of embryonic life by way of the mother-to-child connection, affecting infant brain development and mental health Milani et al., 2017). Studies have demonstrated that the gut microbiota interacts with the CNS through multiple routes, further deepening our understanding of its influence on mental health, including depression (Foster and McVey Neufeld, 2013;Morais et al., 2021;Simpson et al., 2021). Neural signal networks in the brain-gut-microbiota system are important and fast response routes, including the enteric nervous system (ENS), vagus nerves and spinal nerves. The ENS, which is also known as the "brain in the gut" or "second brain," controls the gut's external and internal environment (Ganz, 2021;Margolis et al., 2021;Niesler et al., 2021). The complex regulation of the ENS serves a multifaceted role in modulating microbial composition, microbiota-related metabolites, neurotransmitter and immune signaling, and protection of the intestinal barrier (Margolis et al., 2021;Yoo and Mazmanian, 2017). Thus, bidirectional communication between the CNS and ENS, as well as other signals, forms part of the network for the brain-gut-microbiota axis. The vagal pathway has been repeatedly identified as the most direct link for microflora signals to reach the brain (Morais et al., 2021). On one hand, the vagus nerve is responsible for regulating metabolic homeostasis and feeding behavior, such as gastrointestinal motility and secretion functions (Pavlov and Tracey, 2012). On the other hand, the vagus nerve is involved in the mechanism of the inflammatory actions among the brain, gut, and other organs in the body (Pavlov and Tracey, 2012). Through its afferent and efferent arms, the vagus nerve senses inflammatory cytokines mediated by pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), and then integrates afferent signaling into the brain; it also plays an output function in regulating immune activation and inhibiting pro-inflammatory cytokine release from the gut or other organs by the efferent arm, thereby contributing to the inflammatory reflex (Pavlov and Tracey, 2012). In addition, acetylcholine receptors/ligands mediating the cholinergic anti-inflammatory pathway via the vagus nerve have also been proposed as an important immune modulator in the gut microbiota that communicates with the brain (Bonaz et al., 2018;Pavlov and Tracey, 2012).
The immune signal network in the brain-gut-microbiota system is a complicated bidirectional signaling route with functions that extend beyond neuroinflammation. Activation of the immune system induced by the gut microbiota occurs not only in the intestinal immune system but also through long-distance regulation of brain immune cells Salvo-Romero et al., 2020). Both innate and adaptive immune responses have been associated with the gut microbiota and brain. The innate immune system related to the brain-gut-microbiota axis mainly includes monocytes and macrophages in the peripheral system, glial cells like microglia and astrocyte in the brain, and dendritic cells (DCs) and innate lymphoid cells in the gut (Salvo-Romero et al., 2020;Zheng et al., 2020a). For example, monocytes and dendritic cells can establish interaction signals in the brain-gut-microbiota axis through PRRs that are indirectly activated or directly mediated by gut microbiota or microbiota-related products (Salvo-Romero et al., 2020;Sun et al., 2020a;Zheng et al., 2020a). In the adaptive immune system, Th17 and Treg cells are important immune effectors in the brain-gut-microbiota axis related to microbiome-immunity crosstalk in homeostasis and disease (Salvo-Romero et al., 2020; Zheng et al., 2020a). In addition, the gut barrier and blood-brain barrier (BBB) protect against invasion by external pathogenic microbes or related abnormal signal molecules, which all must be balanced by the innate and adaptive immune system (Margolis et al., 2021).

Findings from rodent models
It is well recognized that stress plays a role in the pathophysiology of depression and that resilience is mediated by adaptive changes in several circuits, including BDNF, pro-inflammatory cytokines, and spleen (Hashimoto, 2019(Hashimoto, , 2020Qu et al., 2017;Yang et al., 2015aYang et al., , 2015bYang et al., , 2016aZhang et al., 2021). Studies have shown that some rodents are resilient to chronic social defeat stress (CSDS) and learned helplessness (LH). Our group reported higher levels of Bifidobacterium in CSDS resilient mice compared with control and CSDS susceptible mice (Yang et al., 2017a). Furthermore, oral administration of Bifidobacterium significantly increased the number of mice resilient to CSDS compared with vehicle treatment (Yang et al., 2017a), suggesting that Bifidobacterium confers resilience to CSDS. We also reported that the relative abundances of the genera Lactobacillus, Clostridium cluster III, and Anaerofustis in LH susceptible rats were significantly higher than those in control and LH resilient rats, and that the levels of acetic acid and propionic acid in the feces of LH susceptible rats were lower than those in control and LH resilient rats (Zhang et al., 2019a). Interestingly, antibiotic-induced microbiome depletion is associated with resilience to CSDS in mice (Wang et al., 2020c). It has also been reported that betaine supplementation contributes to resilience to anhedonia in mice subjected to CSDS through anti-inflammatory actions (Qu et al., 2020). Taken together, these findings support that the brain-gut-microbiota axis plays a role in susceptibility and resilience to stress (Hashimoto, 2020;Wei et al., 2021).
Increasing evidence from preclinical studies suggests that the brain-gut-microbiota axis plays a crucial role in depression-like phenotypes Siopi et al., 2020Siopi et al., , 2017b. Rodents with depression-like behaviors induced by stress have been demonstrated to have abnormal levels of gut microbiota-related SCFAs and other metabolites such as alanine, isoleucine, L-threonine, serine and tyrosine, which may be associated with depressive-like phenotypes and altered levels of 5-HT in the brain (Li et al., 2019a;Wu et al., 2020).
Research demonstrating that administration of microbiota or their metabolites can cause or rescue depression-like phenotypes suggests a bidirectional regulatory effect of the brain-gut-microbiota axis on depression. In some studies, fecal samples containing microbial communities were obtained from patients with depression or mice with depression-like behaviors. Subsequently, fecal microbiota transplantation (FMT) of the "depression-related microbiome" caused depressive-like phenotypes in rodents compared with controls (Knudsen et al., 2021;Wang et al., 2020b). In contrast, treatment with a mixture of SCFAs, including acetate, butyrate, and propionate, has been shown to alleviate stress-induced depressive-like behaviors in mice (van de Wouw et al., 2018).
A recent iTRAQ (Isobaric Tag for Relative and Absolute Quantitation)-based proteomics analysis conducted on a mouse model of depression caused by FMT from depressed patients identified protein alterations in serum, prefrontal cortex, cecum, and liver . The microbiota-driven protein profile was dramatically altered in these tissues, and the altered protein profiles were predicted to play roles in the inflammatory immune response and metabolic regulation, which may contribute to depression via the gut-brain axis . A better understanding of marked alterations from the gut to brain, or from the brain to gut, can strengthen evidence of the role of the brain-gut axis in depression (Jiang et al., 2015;Naseribafrouei et al., 2014;Park et al., 2013;Tillmann et al., 2019;Yang et al., 2020a;Zheng et al., 2016Zheng et al., , 2020b (Fig. 2).

Findings from depressed patients
Numerous clinical studies have reported abnormal composition of the gut microbiota in patients with depression compared to healthy controls, further suggesting a role of the brain-gut-microbiota axis in depression (Jiang et al., 2015;Naseribafrouei et al., 2014;Park et al., 2013;Tillmann et al., 2019;Yang et al., 2020a;Zheng et al., 2016Zheng et al., , 2020b. One such study also found that gut microbiota dysbiosis in depressed patients was associated with reduced BDNF levels and that Faecalibacterium was related to clinician-related depression scale score, contributing to the severity of depressive symptoms (Jiang et al., 2015). A recent study found that gut microbiota DNA contributes to the depression phenotype in distinguishing depressed patients from controls, as shown by machine learning targeting single nucleotide exact amplicon sequence variants in patients' gut microbiome (Stevens et al., 2021). An in-depth study involving depressed patients adopted a multilevel omics perspective to understand alterations in the gut microbiome and metabolomics as contributors to gut ecology in the pathogenesis of depression (Yang et al., 2020a). The overall phenotype of gut ecology, including 3 bacteriophages, 47 bacterial species, and 50 fecal metabolites, in depressed patients differed significantly from that of healthy controls. The altered gut microbiota and metabolites in depressed patients included Blautia sp. Marseille-P2398, Blautia wexlerae, Ruminococcus sp. 5_1_39BFAA, Oscillibacter sp. ER4, phosphate, and L-homoserine, which may be important markers of depression (Yang et al., 2020a). A study of depression and bipolar disorder also noted a distinctly different composition of the gut microbiota in patients compared with healthy controls (Zheng et al., 2020b). Depression was associated with major changes in the family level of Bacteroidaceae, while the family levels of Lachnospiraceae, Prevotellaceae, and Ruminococcaceae mainly contributed to bipolar disorder (Zheng et al., 2020b).
Several systematic reviews and meta-analyses have been conducted to evaluate the evidence of gut microbiota perturbations in patients with depression. One meta-analysis showed that the genera Corprococcus and Faecalibacterium were decreased in depressed patients compared to non-

Fig. 2.
The role of the brain-gut-microbiota axis in depression. An unhealthy lifestyle due to increased and sustained stress, infection, or other factors can cause dysbiosis of the gut microbiota. Abnormal conditions in the body may be bidirectionally regulated by the brain-gut-microbiota axis via neural signals, immune signals, or chemical signal, thereby contributing to depression. Research has demonstrated that fecal microbiota transplantation (FMT), diet, psychobiotics, and antidepressants (e.g., SSRIs, SNRIs, arketamine) can restore abnormalities in the gut microbiota, abnormal brain function, and depressive symptoms via the brain-gut-microbiota axis. CNS: depressed controls, and that depressive symptoms improved in interventional studies with probiotics (Sanada et al., 2020). Another recent meta-analysis showed that gut microbiota perturbations were associated with a transdiagnostic pattern with depletion of certain anti-inflammatory butyrate-producing bacteria and enrichment of pro-inflammatory bacteria in patients with psychiatric disorders including depression (Nikolova et al., 2021). A better understanding of marked alterations from gut to brain or from brain to gut will ascertain the role of the brain-gut axis in patients with depression (Fig. 2).

Role of the vagus nerve in depression
Crosstalk between the brain and gut microbiota is regulated through various routes including the vagus nerve, immune system, and ENS (Bonaz et al., 2018;Cawthon and de La Serre, 2018;Cryan et al., 2019;Forsythe et al., 2014). The vagus nerve acts as a connection among the brain, gut microbiota, and immune system (Liu and Forsythe, 2021). Depression is associated with vagus nerve signaling, which is implicated in inflammatory regulation and modulation by neuroactive molecules (Breit et al., 2018).
In 2011, Bravo et al. (2011) reported that oral gavage with Lactobacillus rhamnosus attenuated stress-induced depression-like behaviors in mice compared with controls, and that vagotomy blocked the effects of Lactobacillus rhamnosus in brain chemistry and depression-like behaviors. We previously reported that subdiaphragmatic vagotomy (SDV) attenuated depression-like behaviors, higher levels of pro-inflammatory cytokines [i.e., interleukin-6 (IL-6) and tumor necrosis factor (TNF-α)], decreased expression of synaptic proteins, and abnormal composition of the gut microbiota in mice after administration of LPS . Furthermore, ingestion of "depression-related" microbes (Lactobacillus intestinalis and Lactobacillus reuteri) led to depression-like phenotypes, higher plasma IL-6 levels, and decreased expression of synaptic proteins in the prefrontal cortex (PFC) of antibiotic-treated mice . Interestingly, SDV blocked the development of depression-like behaviors, elevation of plasma IL-6 levels, and downregulation of synaptic proteins in the PFC after ingestion of these two microbes.
The α7 nicotinic acetylcholine receptor (nAChR) plays a key role in the cholinergic immune system in the brain and periphery (Hashimoto, 2015b;Rosas-Ballina and Tracey, 2009;Wang et al., 2003). We previously reported that α7 nAChR knock-out (KO) resulted in depression-like behaviors via systemic inflammation (Zhang et al., 2016). Another study reported that FMT from α7 nAChR KO mice with depression-like phenotypes caused depression-like phenotypes in antibiotic-treated mice, and that SDV blocked these depression-like behaviors after FMT . Furthermore, we reported that FMT from CSDS susceptible mice caused a depression-like phenotype in antibiotic-treated wildtype mice and resilient Ephx2 KO mice, suggesting that the depression-related microbiome contributes to the conversion of resilient Ephx2 KO mice into KO mice with depression-like phenotypes . SDV was also shown to block the development of depression-like phenotypes after ingestion of depression-related microbiome. Collectively, these findings support that the vagus nerve is responsible for the functional communication of brain-gut-microbiota axis in depression, resulting in depression-like phenotypes.
In contrast, the vagus nerve may not play a role in gut-brain communication during social stress since social activity and corticosterone levels in SDV-operated antibiotic-treated mice were not significantly different than those in sham-operated antibiotic-treated mice . It should be noted that the surgical vagotomy procedure has limitations for differentiating afferent from peripheral signal input to the brain, or efferent from CNS output to a peripheral organ like the gut, thereby contributing to alterations in immune response, brain function, or behavior (Liu and Forsythe, 2021).
Mouse models of depression have been employed to study neuronal activity in response to Lactobacillus rhamnosus (JB-1) treatment. One study found that alterations in c-Fos protein in brain regions were associated with vagal signaling in a dependent or independent manner (Bharwani et al., 2020). Given such findings, there is great interest in identifying the precise route of the vagus nerve to the brain-gut-microbiota axis in depression. Seven subtypes of the vagus motor nerve are connected to different peripheral organs by single nucleus RNA-seq in Cre driver mice (Tao et al., 2021). Studies such as this will contribute to further understanding of what kind of vagal motor neuron subtype plays a key role in depression.

Immune regulation in depression
Within immune regulation through the brain-gut-microbiota axis, numerous studies have focused on alterations in inflammatory cytokines to investigate the immune response in depression. However, there is a complex immune signal network behind such inflammatory changes, including innate and adaptive immune regulation in the gut, brain, and systemic circulation, which has emerged as part of the communication system of the microbiota, gut, and brain in depression Fung, 2020;Morais et al., 2021;Rudzki and Maes, 2020). For example, dysfunction in the gut-microbiota-immune-brain axis may cause brain damage during neurophysiological development in premature neonates (Seki et al., 2021).
One hypothesis is that the microbiota-gut-immune-glia axis plays a role in depression (Rudzki and Maes, 2020). Erny et al. (2015) reported substantial contributions of the host microbiota to microglia homeostasis, demonstrating that germ-free (GF) mice showed global defects in microglia with altered cell proportions and an immature phenotype, leading to impaired innate immune properties. Notably, recolonization with complex microbiota partially restored microglia features and microbiota-derived SCFAs regulated microglia homeostasis. Glial cells, including microglia, astrocytes, oligodendrocytes, and ependymal cells, interact with neurons to influence brain health and disease, such as depression (Rudzki and Maes, 2020). Glial functions may be driven by the gut microbiota via neural and chemical signal routes. The gut microbiota is of significance to microglial activation states from pro-inflammatory to anti-inflammatory, and microglia dysfunction can trigger signaling cascades of neuroinflammation in depression (Carlessi et al., 2021;Fung et al., 2017;Rudzki and Maes, 2020). For example, rifaximin and minocycline are effective in attenuating depressive-like phenotypes induced by stress; these antidepressant-like effects were associated with microbial composition and metabolites, followed by alteration in brain functions, brain microglia (Iba1), and peripheral inflammatory cytokines [e.g., tumor necrosis factor (TNF)-α, interleukin (IL)− 1β, interferon (IFN)-γ, IL-12] Schmidtner et al., 2019). Under different pathological states, the gut microbiota can influence other factorssuch as dietary regulation, SCFAs, tryptophan metabolism, neurotransmitters like GABA and 5-HT, the hypothalamic pituitary adrenal (HPA) axis, and vagus nerve reflexto activate microglia and other glia cells (Abdel-Haq et al., 2019;Rudzki and Maes, 2020). We reported significant correlations between microglial markers in the brain and the relative abundance of several bacteria in PLX5622 (colony-stimulating factor 1 receptor inhibitor)-treated mice, suggesting microbiome-microglia crosstalk via the brain-gut axis . Collectively, these findings suggest that regulation of microglia activation states (M1, M2) via the gut-brain axis may be a promising therapeutic approach for depression (Abdel-Haq et al., 2019;, since depression may be considered as a microglial disease (Yirmiya et al., 2015).
An increasing number of preclinical and clinical studies suggest that Th17 and Treg cells make a significant contribution to depression (Beurel et al., 2013;Beurel and Lowell, 2018;Ghosh et al., 2022;Kim et al., 2021;Li et al., 2010;Poletti et al., 2017;Schiweck et al., 2020;Sun et al., 2020a). Intestinal Th17 and Treg cells are regulated by the gut microbiota, while brain-related Th17 and Treg cells maintain immune homeostasis for controlling neuroinflammation, microglia activation, astrocyte activation, and brain development during pregnancy (Bauer and Teixeira, 2021;Beurel and Lowell, 2018;Carlessi et al., 2021;Osborne et al., 2019;Pandiyan et al., 2019). Notably, an imbalance between Th17 and Treg cells, or the ratio of Th17/Treg cells, has been reported to play a critical role in depression. Th17/Treg cells are specifically required for regulation of the brain-gut-microbiota axis in brain neuroinflammation (Grosse et al., 2016;Hong et al., 2013;Medina-Rodriguez et al., 2020;Schiweck et al., 2020;Westfall et al., 2021a). Imbalance between Th17 and Treg cells regulated by the gut microbiota could confer resilience versus susceptibility to stress (Ambree et al., 2019;Westfall et al., 2021aWestfall et al., , 2021b, and may be involved in the antidepressant effect and side effects of ketamine (Cui et al., 2021). Specific gut microbiota, such as segmented filamentous bacteria, Clostridia, Bacteroides fragilis, Lactobacillus reuteri, and Bifidobacterium, as well as their metabolites like SCFAs and ATP, are also involved in Th17/Treg activity, thereby shaping the imbalance between Th17 and Treg cells (Cheng et al., 2019;Zhu et al., 2020). Further studies are needed to identify the interrelationship between the gut microbiota and Th17/Treg cells in depression to understand the brain-gut-microbiota axis in the immune signal network.

SCFAs and D-amino acids
SCFAs have become one of the most studied classes of small molecules in recent years due to their essential role in gut microbiota metabolism. In addition to maintaining intestinal homeostasis (van der Hee and Wells, 2021), SCFAs can shed light on why dysbiosis in the gut microbiota is involved in the pathophysiology of depression via brain-gut signals (Dalile et al., 2019;Silva et al., 2020;Wu et al., 2020). For example, the levels of acetic acid, propionic acid, and pentanoic acid are altered in the chronic restraint stress (CRS) model of depression, and mice with a depression-like phenotype show a significant decrease associated with gut microbiota dysbiosis and neurotransmitters such as 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) compared with controls (Wu et al., 2020). A comparison study of depressed patients and healthy controls found that depressed patients showed decreased butyrate (anti-inflammatory properties) produced by Faecalibacterium and increased LPS (pro-inflammatory properties) produced by Flavonifractor (Liu et al., 2020).
A recent systematic review of 26 studies of anxiety disorder and depression concluded that pro-inflammatory species, such as Enterobacteriaceae and Desulfovibrio, are higher whereas SCFAs related to Faecalibacterium are lower in patients compared with controls (Simpson et al., 2021). It has been suggested that butyrate and other SCFAs may improve depression-like behaviors through their anti-inflammatory effects, inducing histone hyperacetylation and elevating BDNF levels (Stilling et al., 2016).
SCFAs can regulate BBB permeability and cross the BBB, thereby affecting neurons and immune cell crosstalk that contributes to brain function and behavior (Agirman and Hsiao, 2021;Dalile et al., 2019;Silva et al., 2020;Xiao et al., 2020). Treatment with a mixture of acetic acid, propionic acid, and butyric acid improved permeability of the intestinal barrier induced by chronic stress (van de Wouw et al., 2018). Non-absorbable antibiotic rifaximin targeting the brain-gut-microbiota axis was shown to improve depressive-like behavior caused by chronic unpredictable mild stress (CUMS) . The antidepressant-like effect of rifaximin was reflected by inhibition of microglia activation in phagocytosis and increase of butyric acid levels in the brain, which were associated with the gut microbiota taxa Ruminococcus bromii and Lachnospiraceae . Butyrate treatment also ameliorated LPS-induced depression-like phenotype through anti-inflammation (Yamawaki et al., 2018). Understanding the neuroactive potential of SCFAs involved in host health and disease may involve other routes targeting the brain-gut-microbiota axis. For example, SCFA-induced depression may be related to Th17/Treg cells immune regulation (Westfall et al., 2021b), TLR4/NF-κB-mediated inflammatory response (Sun et al., 2020b), tryptophan catabolites metabolism , and the endocannabinoid system Vijay et al., 2021). Taken together, these studies support that SCFAs play various crucial roles in the brain-gut-microbiota axis and may be a novel therapeutic target for depression (Merchak and Gaultier, 2020).
At present, the precise mechanisms underlying the role of the brain-gut-microbiota axis in depression are unclear. It is difficult to definitively show how many or what key routes are responsible for the pathophysiology of depression. Nevertheless, the brain-gut-microbiota axis is a promising therapeutic target for depression.
It has been suggested that gut microbiome can produce free D-amino acids such as D-serine and D-glutamate Matsumoto et al., 2018;Nakade et al., 2018;Sasabe and Suzuki, 2018). A study using high-sensitivity liquid chromatography-tandem mass spectrometry showed that 12 free D-amino acids (D-alanine, D-arginine, D-aspartate, D-glutamine, D-glutamate, D-allo-isoleucine, D-leucine, D-lysine, D-methionine, D-phenylalanine, D-serine, D-tryptophan) could be produced by intestinal microbiota (Matsumoto et al., 2018). Accumulating evidence suggests that D-amino acids such as D-serine and D-glutamate might play a role in the pathophysiology of a number of neuropsychiatric disorders including depression, schizophrenia, and Alzheimer's disease Dong et al., 2018;Fujita et al., 2016;Hashimoto et al., 2003Hashimoto et al., , 2004Hashimoto et al., , 2005Hashimoto et al., , 2016Yamada et al., 2005). However, the precise roles of D-amino acids and gut microbiota in neuropsychiatric disorders such as depression are unknown. Further detail study is needed to investigate how gut microbiome-derived D-amino acids play a role in health and diseases in the host.

FMT
A brief review of the history of FMT is helpful for understanding the implications of the brain-gut-microbiota axis in depression. A milestone study by Sudo et al. (2004) compared the brain-gut-microbiota axis among GF, specific pathogen free (SPF,) and gnotobiotic mice, reporting that GF mice had a high level of plasma adrenocorticotropic hormone (ACTH) and corticosterone, and a low level of BDNF in brain regions, within a model of HPA response to restraint stress. In contrast, Bifidobacterium (B.) infantis or colonizing microbes with SPF mice feces showed a reversal effect in response to HPA stress (Sudo et al., 2004). Several years later, Bercik et al. (2011) reported that the gut microbiota affected brain BDNF levels and exploratory behavior in antibiotic-induced dysbiosis model mice, and that these alterations were reversed following withdrawal of antimicrobials. Importantly, the authors also demonstrated that the brain-gut-microbiota axis was regulated by FMT: the gut microbiota of NIH (National Institute of Health) Swiss mice colonized inside GF BALB/c mice increased brain BDNF level and exploratory behavior, while the gut microbiota of BALB/c mice colonized inside GF NIH Swiss mice decreased exploratory behavior (Bercik et al., 2011). Fei and Zhao (2013) reported that disease-related phenotypes can be transmitted from one individual to another by FMT, such that recipient mice displayed phenotypes of obesity and insulin resistance after being transplanted with the gut microbiota of Enterobacter from morbidly obese humans, accompanied by serum endotoxin and inflammatory cytokines increases. A randomized controlled trial showed that intestinal flora from lean donors restored insulin sensitivity and increased bacterial diversity in obese patients with metabolic syndrome, and that this effect may be regulated by microbiota-derived butyrate (Vrieze et al., 2012). These findings strongly suggest that microbiota have systemic functions in transferring the host phenotypes to other individuals via the whole microbiota, specific microorganisms, or microbiota-related metabolites (Koh and Backhed, 2020).
Reconstitution with microbes or FMT may be an effective intervention for various brain diseases. A review article highlighted the utility of FMT as a therapeutic option for depression (Koh and Backhed, 2020). As detailed above, depressive-like phenotypes can be transferred from mice with depression-like phenotypes or depressed patients via FMT, suggesting that FMT may have beneficial effects for improving depression (Kelly et al., 2016;Knudsen et al., 2021;Li et al., 2019c;Marcondes Avila et al., 2020;Pu et al., 2021;Rao et al., 2021aRao et al., , 2021bWang et al., 2020a;Wang et al., 2020b;Zhang et al., 2019b;Zheng et al., 2016). Various case reports, meta-analyses, and systematic reviews have also showed that FMT improves depressive symptoms in patients with depression (Cai et al., 2019;Chinna Meyyappan et al., 2020;Cooke et al., 2021;Evrensel and Tarhan, 2021;Fond et al., 2020;Green et al., 2021;Hinton, 2020;Settanni et al., 2021;Xu et al., 2021). However, it is difficult to draw definitive conclusions from FMT clinical trials of patients with depression because short-term and long-term adverse events of FMT cannot be avoided or determined in the context of current FMT therapeutic strategies (Baunwall et al., 2021;Merrick et al., 2020).

Diet for depression
Epidemiological evidence suggests that the effects of diet on depression may be relevant to the stage of life including prenatal depression (Marx et al., 2019). Systematic reviews and meta-analyses have concluded that healthy dietary interventions have beneficial effects on both prevention and improvement in depression (Lassale et al., 2019;Wu et al., 2021a). A large-scale metagenomics study of 1054 volunteers confirmed that there is a close relationship among intestinal microbes, dietary health, and depression (Valles-Colomer et al., 2019). Butyric acid-producing Faecalibacterium and Coprococcus bacteria were identified as important indicators of a high-quality diet, even in patients with depression treated with antidepressants (Valles-Colomer et al., 2019). Using machine learning strategies in the American Gut Project, host variables such as diet were identified as important variables contributing to the human gut microbiota (Vujkovic-Cvijin et al., 2020). In light of these findings, a low-quality diet is thought to trigger a series of reactions that include changing intestinal microbiota diversity, reducing essential nutrient intake, reducing the growth substrates of specific microorganisms, and further aggravating dysbiosis of gut microbial communities in patients with depression .
A Western diet high in saturated fat and sugars has been shown to have a deleterious impact on neuropsychological outcomes, increase endotoxin production from commensal bacteria, and promote neuroinflammation and BBB leakage, thereby leading to cognitive decline and depression Noble et al., 2017;Psaltopoulou et al., 2013). A high fructose diet treatment in mice lasting for several weeks led to neuroinflammatory alteration in the hippocampus, mediated by the NLRP6 inflammasome impairing the intestinal epithelial barrier, and SCFA supplementation ameliorated neuroinflammation caused by this high fructose diet . Adult rats treated with a high-fat diet for 3 weeks showed depression-like behavior and a low level of BDNF-TrkB signaling in brain regions (frontal cortex and hippocampus), although there were no data indicating gut microbiota dysfunction (Sharma et al., 2012). The ability of a high-fat diet to induce a depression-like phenotype may relate to hypothalamic PKA (protein kinase A) signaling and leptin/LepRb (leptin receptor) signaling (Vagena et al., 2019;Yang et al., 2016b). Negative emotions can affect our eating behavior and energy intake by promoting high calorie food intake and hedonistic eating, which are related to neuropeptide Y in the brain (Herzog, 2020). In contrast, healthy eating patterns like the Mediterranean diet have been shown to increase gut microbial taxa and reduce inflammation markers, thus contributing to improvement in depressive symptoms (Firth et al., 2020). In addition, Norwegian and Japanese diets; omega-3 polyunsaturated fatty acids (PUFAs) enriched foods; and healthy dietary patterns involving a combination of high intake of whole grains, olive oil, vegetables, fruits, fish, soy, and lower fat dairy may be associated with a lower risk of depression (Deacon et al., 2017;Lassale et al., 2019;Nishi et al., 2020;Okubo et al., 2019;Opie et al., 2017). Given such findings, diets targeting the brain-gut-microbiota axis have been suggested to benefit the gut microbiota, promote a healthier microbiome, maintain the integrity of the intestinal barrier, and contribute to brain development (Lassale et al., 2019;Silva et al., 2020;Zhang and Kutateladze, 2018;Zheng et al., 2020a).

Psychobiotics for depression
Given accumulating evidence for the existence of the brain-gut-microbiota axis and its potential implications in psychiatric disorders, the concept of psychobiotics was proposed in 2013. Psychobiotics are considered a class of probiotic that can benefit people suffering from psychiatric disorders, including depression . With an expanded scope of probiotic function, psychobiotics mainly include probiotics with adequate amounts of beneficial bacteria and prebiotics with the ability to be utilized by gut microflora to convert beneficial substances (Cunningham et al., 2021;Gibson et al., 2017;Sarkar et al., 2016). Psychobiotics targeting the bacteria-gut-brain signals likely involve multiple routes and neural signal networks, such as the ENS and vagus nerve, immune signal network of interactions between microbe-associated molecular patterns and PRRs like TLRs, C-type lectins and inflammasomes, and gut microbiota-derived SCFAs (Sarkar et al., 2016). A recent review provides a good summary of the source of probiotics and prebiotics (Cunningham et al., 2021). Probiotics principally include human microbiota, fermented foods, environment-related soil, plants, and animals, whereas prebiotics mainly comprise plants, fungi, animals, microbes, and chemical and physical modification (Cunningham et al., 2021). For example, fructo-oligosaccharides and galacto-oligosaccharides are regarded as prebiotics that exhibit antidepressant and anti-inflammation effects in the chronic stress-induced mouse model of depression (Burokas et al., 2017).
Furthermore, systematic reviews and meta-analysis suggest that psychobiotics have anxiolytic and antidepressant effects (Chudzik et al., 2021;Cohen Kadosh et al., 2021;Liu et al., 2019;Smith et al., 2021). Animal studies have confirmed these effects while simultaneously exploring the mechanisms underlying the antidepressant effects of psychobiotics. For example, strain MCC1848 from heat-killed Lactobacillus helveticus attenuated depressive-like behaviors induced by subchronic and mild social defeat stress (Maehata et al., 2019). In CUMS model animals, Bifidobacterium breve CCFM1025 had antidepressant-like effects, as evidenced by improvement in depression-like behavior, reshaping of the gut microbiota community, reduced inflammation, and balancing of HPA axis hyperfunction . In addition, CCFM1025 upregulated the BDNF level while downregulating pCREB (cAMP response element-binding protein)-c-Fos signaling in the PFC in response to stress, followed by increased levels of SCFAs and 5-hydroxytryptophan . Recent reviews and studies on the beneficial effects of psychobiotics for depression have mainly concentrated on the genera Lactobacillus and Bifidobacterium, finding that different strains have specific contributions to mental health ( Barros--Santos et al., 2020;Burnet and Cowen, 2013;Duranti et al., 2020;Misra and Mohanty, 2019;Sarkar et al., 2016). Other psychobiotics have also been found to improve depression. In a CRS mouse model of depression, Akkermansia muciniphila was found to ameliorate the depressive-like phenotype by restoring the abnormal gut microbiota and metabolites and by regulating corticosterone, dopamine, and BDNF levels (Ding et al., 2021). Notably, several studies have reported null results for psychobiotics, reporting no improvement in depressive-like behavior Vaghef-Mehrabany et al., 2020). As a result, it is currently difficult to conclude whether psychobiotics have antidepressant effects due to their various modes of action and the possibility of strain-specific effects, multi-strain formulations, or other modalities (Bambury et al., 2018;Vaghef-Mehrabany et al., 2020). Nevertheless, plant-based psychobiotics targeting the brain-gut-microbiota pathway have been developed and hold high promise as novel antidepressants Vaghef-Mehrabany et al., 2020). Overall, further studies are needed to confirm the beneficial effects of psychobiotics in depression.

Antidepressants
Antidepressants, such as SNRIs and SSRIs, are the most commonly prescribed drugs for depression despite various side effects, delayed clinical onset, and high non-response rates (Galecki et al., 2018;Hoyer, 2020;Wei et al., 2021). Although the detailed biological mechanisms underlying antidepressant actions remain unclear (Casarotto et al., 2021), recognition of the role of intestinal microbes may be a turning point (Klunemann et al., 2021). An abnormal composition of the gut microbiota is associated with depression (Foster and McVey Neufeld, 2013;Jiang et al., 2015), and antidepressants have anti-microbial and anti-inflammatory effects related to gut microbiota ecological restoration (Galecki et al., 2018;Hashimoto, 2015a;Macedo et al., 2017;McGovern et al., 2019). Various preclinical studies have suggested that SSRIs and other kinds of antidepressants exert anti-microbial activity, and that treatment efficiency may be regulated or governed by microorganisms at multiple taxonomic levels (Ait Chait et al., 2020;Dethloff et al., 2020;Lukic et al., 2019;McVey Neufeld et al., 2019;Ramsteijn et al., 2020;Shen et al., 2021). For example, Duan et al. (2021) reported that the gut microbiota community and metabolites differed significantly between responders and non-responders treated with escitalopram in a CUMS model of depression, with Prevotellaceae_UCG-003 increasing and Ruminococcaceae and Lactobacillaceae decreasing in the responder group compared with the non-responder group. Using the same depression model, the antidepressant-like effects of fluoxetine had strong correlations with the gut microbiota, and its antidepressant efficacy was weakened by decreasing 5-HT biosynthesis and disrupting tryptophan metabolism via the gut microbiota Sun et al., 2019). Thus, targeting the brain-gut-microbiota axis could be integrated with antidepressants to achieve better understanding of the neurobiological processes underlying depression Harmer et al., 2017;Siopi et al., 2020).
The rapid antidepressant effects of ketamine may also be correlated with the gut microbiota (Hashimoto, 2019(Hashimoto, , 2020Wang et al., 2020d;Wei et al., 2021;Wilkowska et al., 2021). (R)-ketamine (arketamine) has more potent and longer-lasting antidepressant effects with fewer side effects in rodents and humans compared to (S)-ketamine (esketamine) (Bonaventura et al., 2021;Chang et al., 2019;Leal et al., 2021;Yang et al., 2015c;Yao et al., 2021;Zhang et al., 2014Zhang et al., , 2020b. However, the precise molecular mechanisms underlying the differential activities of arketamine and esketamine are yet to be fully understood (Hashimoto, 2019(Hashimoto, , 2020Heifets et al., 2021;Wei et al., 2020Wei et al., , 2021Yao et al., 2021). Interestingly, preclinical data suggest that (R,S)-ketamine or (R)-ketamine has the ability to ameliorate the abnormal composition of gut microbiota associated with depression-like behaviors (Getachew et al., 2018;Huang et al., 2019;Qu et al., 2017;Yang et al., 2017b). The potential role of the brain-gut-microbiota axis in the antidepressant-like action of arketamine is of great interest. In contrast to esketamine, arketamine has specific effects on Mollicutes and Butyricimonas at different taxonomic levels of the gut microbiota, which are associated with its robust antidepressant-like actions in CSDS susceptible mice (Yang et al., 2017b). In contrast to antidepressant candidate lanicemine, arketamine showed significant antidepressant-like effects in CSDS susceptible mice (Qu et al., 2017). The reason for this difference may be partially due to the restoration of decreased order levels of Bacteroidales and Clostridiales. The antidepressant-like actions of arketamine may also be associated with gut microbiota alterations at different taxonomic levels, including the family levels of Ruminococcaceae and Mogibacteriaceae and the genus level of Clostridium. Thus, the brain-gut-microbiota axis may be involved in the antidepressant-like effects of (R,S)-ketamine and arketamine (Hashimoto, 2019(Hashimoto, , 2020Wei et al., 2021;Wilkowska et al., 2021), although more detailed studies are needed.

Conclusions and perspectives
The brain-gut-microbiota axis is a complex and interactive system that includes a neural signal network, immune signal network, and chemical signal network. All of these networks appear to be involved in the pathophysiology of depression. In particular, the vagus nerve, Th17/ Treg activity, microglia, gut microbial composition, and SCFAs have attracted increasing research focus for understanding how the brain-gut-microbiota axis contributes to depression (Fig. 2). The brain-gut-microbiota axis is considered a new paradigm with the potential to provide new treatment strategies for depression (Bambury et al., 2018;Wei et al., 2021). Beneficial effects of diets and psychobiotics targeting the brain-gut-microbiota axis in depression have been increasingly reported in both preclinical and clinical studies (Bambury et al., 2018;Long-Smith et al., 2020;Marx et al., 2021;Simpson et al., 2021). However, the precise mechanisms underlying the role of the brain-gut-microbiota axis in depression remain unclear. Further understanding of the role of the brain-gut-microbiota axis in depression will provide new therapeutic approaches for supporting mental health.

Acknowledgements
This study was supported by the grants from Japan Society for the Promotion of Science (to K.H., 21H00184, 21H05612, and 21H02846).

Conflict of interest statement
Dr. Hashimoto is the inventor of filed patent applications on "The use of R-Ketamine in the treatment of psychiatric diseases", "(S)-norketamine and salt thereof as pharmaceutical", "R-Ketamine and derivative thereof as prophylactic or therapeutic agent for neurodegeneration disease or recognition function disorder", "Preventive or therapeutic agent and pharmaceutical composition for inflammatory diseases or bone diseases", "R-Ketamine and its derivatives as a preventive or therapeutic agent for a neurodevelopmental disorder", and "Preventive or therapeutic agent and pharmaceutical composition for inflammatory diseases" by the Chiba University. Dr. Hashimoto also declares that he has received research support and consultant from Dainippon Sumitomo, Otsuka, Taisho, Murakami Farm, and Perception Neuroscience. The other authors have no conflict of interest.