Tryptophan and Its Metabolite Serotonin Impact Metabolic and Mental Disorders via the Brain–Gut–Microbiome Axis: A Focus on Sex Differences

The brain–gut–microbiome axis is a complex, bidirectional communication network that enables continuous crosstalk among the brain, gut, resident gut microbiota, and microbial metabolites. This communication occurs through a combination of neural and circulating signals [3,4,5]. Exciting emerging research suggests that gut microbial influence plays a fundamental role in regulating behavior and metabolism, with the brain–gut–microbiome axis being essential for maintaining homeostasis under both healthy and disease conditions [5,6,7,8,9,10,11].

The microbiome consists of all microorganisms residing in and on the body, including fungi, protozoa, archaea, viruses, and bacteria, with the gastrointestinal tract hosting the largest microbial collection. The gut microbiome encompasses all these microorganisms that reside within the gut, their genetic repertoire (including number, classes, variability, and diversity of genes), and the metabolites they produce. Acting symbiotically with the host, the gut microbiome ferments dietary fibers, aids in digestion and absorption, and protects against pathogens. The gut microbiome is influenced by multiple factors, including the host’s genetics, age, and sex [12,13,14,15,16,17,18,19,20]; lifestyle (such as diet, alcohol, exercise, and sleep (e.g., [21])); and chronic diseases (Figure 1). The gut microbiota is also heterogeneous across the gastrointestinal tract, with varying bacterial communities from the oral region to the distal colon, as well as between the mucosal and the luminal layer [22,23]. Microbial composition changes over different life stages within individuals, especially during key transitional periods of brain development and maturation. In early life, microbial composition and metabolites change in parallel to meet the dynamic metabolic demands of growing brains [24]. Moreover, diet significantly impacts gut microbiota composition, which affects food digestion, subsequent energy harvest, and immune development-related epithelial health and homeostasis [25]. For instance, although Firmicutes, Bacteroidetes, Actinobacteria, and Verrucomicrobia are typical phyla in healthy adult microbiota, the ratio of Firmicutes to Bacteroidetes (i.e., Firmicutes/Bacteroidetes) varies widely across individuals [26,27].

With the spread of the Western diet, obesity incidence has been increasing worldwide over the past few decades. Obesity is a key risk factor for low-grade chronic inflammation, which is a likely contributing factor to various complications, including colorectal cancer, as well as type 2 diabetes and cardiovascular disease [28]. Markedly different microbiota profiles exist between lean and obese individuals [29,30]. Manipulating gut microbiota composition with probiotics and prebiotics may improve weight management and fat deposition. High-fat diet (HFD) feeding has been shown to induce epigenomic changes, including histone modifications, in both humans and animal models [31]. Understanding how the host gut microbiome and epigenome respond to diet could provide mechanistic insights into diseases associated with obesity. For instance, probiotic supplementation with Lactobacillus and Bifidobacterium in an animal model of HFD-fed mice has led to reduced weight gain and better metabolic outcomes, such as improved glucose homeostasis and insulin sensitivity [32]. Clinical studies suggest that microbiota modifications, such as receiving gut microbiota from lean donors, may ameliorate symptoms associated with obesity and improve insulin sensitivity in individuals with metabolic syndrome [33]. Therefore, the interaction between gut microbiota and hosts involves a wide range of pathways and presents a promising avenue that affects health and disease.

The brain significantly influences gut motility, permeability, secretion, immune functions, gut microbial gene expression, and the structure and function of the microbiome. This interplay between the brain and the gastrointestinal tract relies on both neural and circulating signals to modulate metabolic and immune functions [34,35,36] (Figure 2).

Using a metagenomic approach, it has been estimated that the gut microbiome contains microorganisms at levels 10-fold greater than all the cells in the human body and has genes that are 150 times more abundant than the human genome [65,66,67]. This vast genetic diversity of the gut microbiome offers a wide range of metabolic, nutritional, hormonal, neural, and immune signals for crosstalk between the host and the gut. These signals directly and indirectly affect local metabolite production, control metabolic functions, regulate immune responses, and modulate behaviors, thus mutually achieving the maintenance of host homeostasis [68,69].

Several experimental strategies are available to researchers in this field. First, changes in tryptophan levels through depletion, supplementation, or molecular manipulation, either acutely or chronically, modify the peripheral and central levels of tryptophan metabolites. For example, preclinical and clinical studies employing these procedures have reported that low levels of the tryptophan metabolite 5-HT are associated with dysregulated eating behavior in animal models and eating disorders in humans, establishing the role of 5-HT in regulating feeding within the CNS. Second, accumulating evidence from animal models manipulating gut microbiota composition—through germ-free animals devoid of microorganisms, antibiotic-treated animals with microbiota deficiency, or animals rich in microorganisms via probiotic supplementation, fecal transplantation, or deliberate infections—suggests that the gut microbiome significantly impacts behavior and metabolism in lab animals, contributing to psychiatric and metabolic disorders seen in humans [11].

These strategies, through rigorous assessment using specific pathogen-free mice and germ-free mice, have illuminated the integrity of the gut microbiota in the functions of tryptophan metabolites, enhanced our understanding of the intricate relationship between the gut microbiome and the brain, and highlighted the crucial role of microbial communities in mediating various functions, including neural activity, metabolism, and behavior [70,71,72].

The biochemical cascades for tryptophan metabolism and tryptophan metabolite synthesis are intricately linked to the microbiota–gut–brain axis, with microbial regulation playing a significant role [79]. Dietary tryptophan, once absorbed through the intestinal epithelium, exists in both free and albumin-bound forms, either remaining in the gut or entering the bloodstream [78]. In the blood, tryptophan levels are influenced by dietary protein intake [80]. The free form of circulating tryptophan serves as the primary substrate for protein synthesis [77].

In the gut lumen, five microbial phyla—Actinobacteria, Firmicutes, Bacteroidetes, Proteobacteria, and Fusobacteria—metabolize tryptophan through various metabolic pathways [81,82]. Tryptophan transporters facilitate its uptake from the gut lumen into bacterial cells, allowing bacteria to sequester it from the host. Most tryptophan metabolism by the microbiota occurs in the distal colon, where bacterial proteolytic activity is the highest [78,83]. Some metabolism by Lactobacilli also occurs in more rostral regions such as the stomach and ileum [82].

Bacteria produce a few neuroactive tryptophan metabolites [84,85]. Around 90% of tryptophan is metabolized and gives origin to kynurenine and its derivatives kynurenic acid and quinolinic acid. Only about 3% is hydroxylated to intermediate 5-hydroxytryptophan (5-HTP), by the rate-limiting enzyme tryptophan hydroxylase (TPH), which is then converted to 5-HT via amino acid decarboxylase. Other pathways yield tryptamine and various indole derivatives, including indole, indole acetic acid (IAA), indole propionic acid (IPA), indole lactic acid, indole acrylic acid, and skatole [85,86].

It is noteworthy that, while this review emphasizes 5-HT, many other tryptophan metabolites produced by the gut microbiota and the CNS act as essential neuroactive molecules and participate in brain–gut communication to regulate energy metabolism, mood, and behavior in humans and lab animal models [1,78,79,82,83,87,88,89,90,91]. For example, the central administration of quinolinic acid, a metabolite from the kynurenine pathway, reduces grooming behavior and shortens the duration of social interaction in mice [91], suggesting anxiogenic effects of quinolinic acid. Findings from animal studies are supported by clinical research. In humans, depression, anxiety symptoms, and cognitive impairments in patients with schizophrenia are associated with the increased production of tryptophan metabolites [92]. Additionally, in women, depression and anxiety related to body image satisfaction are linked to the activation of tryptophan catabolism and CNS availability of quinolinic acid [93]. In both preclinical animal studies and clinical human studies, increased immune responses enhance tryptophan catabolism, resulting in decreased levels of tryptophan and 5-HT, while increasing the production of neurotoxic tryptophan catabolites such as kynurenine and quinolinic acid [94,95]. This shift can lead to heightened depression and anxiety behaviors in both humans and rodents [80,96,97].

5-HT is the most studied neuroactive molecule among all tryptophan metabolites synthesized in both the gut and the brain. Serotonergic neurotransmission also occurs in both the gut and the brain. The majority of 5-HT is produced from tryptophan by the microbiome in the enterochromaffin cells (a subtype of enteroendocrine cells) of the gut [98]. Fluctuations in local 5-HT concentration can influence enteric neural processes and subsequently relay signals along the brain–gut axis, affecting CNS neurotransmission, as has been tested in human biopsy transverse colons and in colons from laboratory rodent [41,99]. Some tryptophan crosses the blood–brain barrier via large amino acid transporters to synthesize 5-HT in the CNS [79]. The experimental manipulation of tryptophan levels modified 5-HT levels in both the gut and the CNS [79].

Two well-characterized homologous isoenzymes of TPH, TPH1 and TPH2, are primarily expressed in the gut and in the CNS, respectively [100]. In humans, circulating tryptophan is used to synthesize 5-HT in the CNS via TPH2 [101,102]. It is noteworthy that, while circulating tryptophan concentration influences CNS 5-HT levels, it is not the sole factor. In clinical studies, other factors have been identified in humans. For example, competition with other amino acids for transport across the blood–brain barrier also plays a critical role [103], and the ratio of tryptophan to other amino acids also determines CNS 5-HT levels [80]. Physiological factors such as exercise can further influence tryptophan distribution and utilization in the CNS in humans [104].

Research supports the importance of gut microbiota in 5-HT synthesis. In humans, approximately 95% of the body’s 5-HT is secreted by mucosal enterochromaffin cells, regulated by microbiota. Certain gut bacteria, including Enterococcus, Pseudomonas, Streptococcus, and Escherichia, can produce 5-HT in tryptophan-rich media [11,86,105,106] by controlling amine transport, metabolism, and biosynthesis [107,108,109]. Additionally, short-chain fatty acids (SCFAs) and particularly butyrate, a metabolite produced by gut microbiota, have complex effects on 5-HT synthesis from human intestinal enterochromaffin cells. Low levels of butyrate promote TPH1 expression and increase 5-HT synthesis, while high levels of butyrate suppress TPH1 expression and decrease 5-HT synthesis [107].

Animal studies also indicate that gut microbiota significantly modulate 5-HT synthesis. Germ-free mice exhibit significantly lower 5-HT levels compared to conventionally housed mice, while the fecal flora colonization of germ-free mice with specific pathogen-free mouse microbiota rapidly increased colonic 5-HT levels within days [108]. Similarly, germ-free mice with human gut microbiota colonization increase their colonic TPH1 mRNA and protein levels, which consequently increases colonic 5-HT levels [107]. Pathogenic bacteria such as Escherichia coli, Vibrio cholerae, and Salmonella typhimurium have been associated with upregulated 5-HT secretion into the lamina propria of the gut [110]. These studies suggest that gut microbiota promote 5-HT production in the gut lumen.

Diet also influences both local gut processes and systemic effects through bacterial structural components and microbial metabolites [86]. The gut microbiome produces many bioactive compounds affecting host physiology, such as SCFAs such as acetate, propionate, and butyrate [111] as well as choline metabolites and lipids [25]. SCFAs can rapidly move from the colonic lumen into colonic epithelial cells and provide a preferred energy source for the epithelia [111]. Some gut bacterial metabolites regulate 5-HT synthesis, and some are substrates for 5-HT, which can further regulate gut functions. For instance, in both mice and humans, metabolites from Clostridium species can stimulate 5-HT synthesis and release from colonic enterochromaffin cells, enhancing gastrointestinal motility [112] and affecting neurotransmitter and hormone activities [113,114].

Research has shown that butyrate levels are lower in both male and female mice fed a high-fat diet (HFD) compared to those on a low-fat diet (LFD). Notably, female mice exhibit more modest decreases in butyrate levels, while male mice show more pronounced reductions [29]. Consequently, HFD-fed mice, particularly males, have higher levels of gut-synthesized 5-HT compared to their LFD-fed counterparts [29].

In humans, lipopolysaccharides (LPSs) stimulated the release of 5-HT from enterochromaffin cells isolated from patients with Crohn’s disease [115]. This regulation of 5-HT synthesis and release from intestinal enterochromaffin cells by gut microbial metabolites represents a form of local paracrine regulation. These metabolites activate receptors located either on afferent nerve terminals near the enterochromaffin cells, triggering enteric reflexes, or directly on the enterochromaffin cells themselves [41,116].

To summarize, microbial products play critical roles in maintaining gut homeostasis. They facilitate the secretion of various enteric neuropeptides and cytokines from mucosal enteroendocrine and immune cells [82]. Additionally, they regulate the biosynthesis and release of various neurotransmitters and neuromodulators by enterochromaffin cells [84,117]. The released enteric neuropeptides, cytokines, neurotransmitters, and neuromodulators, along with microbial metabolites, diffuse through the lamina propria, accessing local receptors or entering the bloodstream, to affect local ENS neurons and remotely signal CNS neurons via vagal innervation [118,119,120]. Ultimately, these interactions help to regulate gut and CNS functions, including appetite and satiety, nutrient metabolism, energy homeostasis, immune responses, mood, and behavior [3,7,9,11,69,121]. In contrast, an HFD impacts the synthesis of SCFAs, which can regulate TPH1 levels and thus affect 5-HT synthesis [29]. The resulting imbalance in SCFA production may lead to the dysregulation of gut–brain communication and mood disorders, highlighting the importance of diet in shaping gut microbiota and their implications for mental health.

Research indicates significant sex differences in tryptophan metabolism, likely influenced by sex hormones. In women, reduced tryptophan and 5-HT synthesis, along with the increased production of neurotoxic and anxiogenic tryptophan metabolites, particularly via the kynurenine pathway, are associated with eating disorders and mood disorders, such as depression and anxiety. Women, regardless of ethnicity, generally have higher levels of free tryptophan but lower total tryptophan levels in plasma compared to men [122]. Additionally, the kynurenine pathway differs between sexes. For example, African and Hispanic American women exhibit lower levels of tryptophan metabolites than their male counterparts [122]. Ethnicity also plays a role in tryptophan metabolism, as Caucasian women, in particular, show the lowest levels of kynurenic acid, which potentially correlates with a lower incidence of schizophrenia in the Caucasian female population [122]. Furthermore, dietary factors that induce acute tryptophan depletion can trigger increased stress-induced anxiety responses in women, such as during a simulated public speaking test [123], suggesting that women may be more sensitive to diminished 5-HT neurotransmission than men. These findings highlight the potentially significant roles of both sex and ethnicity in the complex relationship between tryptophan metabolism and mental health.

Tryptophan metabolism is also regulated by stress and immune responses. For example, in men and women with similar post-traumatic stress disorder severity, the level of indole metabolite (indole-3- propionic acid) decreases in women, but not in men, possibly due to gut microbiota dysbiosis [21]. Other studies have reported that increased levels of gut bacteria-derived LPSs can activate the kynurenine pathway, leading to elevated levels of the neurotoxic quinolinic acid [124]. This pathway activation is more pronounced in women than in men following immune challenges [80], which drives tryptophan metabolism and conversion into the tryptophan metabolite kynurenine, leading to increased kynurenine levels and decreased CNS 5-HT synthesis [124].

Such changes are not only more pronounced in women than in men, but also exacerbated during pregnancy and the menstrual cycle and commonly observed in women during the perinatal period, including at the end of pregnancy, during early postpartum, and in the premenstrual phase. These changes are associated with the heightened symptoms of depression and anxiety observed in pregnant and parturient women [94,124,125,126]. The dynamic fluctuations in sex hormones during pregnancy and the puerperium may enhance kynurenine pathway activity, increasing the risk of anxiety and depression during these life stages. However, one clinical study did not find a correlation between increased kynurenine levels and prenatal or postpartum depression, or at the end of term pregnancy [126]. This discrepancy may be due to the complex interactions between immune responses, stress, and tryptophan metabolism, especially during life stages related to pregnancy and menstrual cycles with dynamic physiological and behavioral changes [126,127,128,129].

Both the sex hormones estrogen and progesterone contribute to enhanced 5-HT synthesis by promoting the expression and activation of key enzymes, such as protein kinase A [130] and, subsequently, phosphorylated TPH [131]. Specifically, estradiol increases TPH mRNA and protein levels without affecting 5-HT content in mice, and progesterone may activate protein kinase A [132]. In guinea pigs, when treated with combined estrogen and progesterone, both TPH and 5-HT levels in the dorsal raphe nucleus (DRN) and the hypothalamus are increased [133]. In ovariectomized (OVX) rats, a rodent model of postmenopause, estradiol treatment alone, but not combined estrogen and progesterone treatment, increased TRH gene expression in the DRN [134]. It is possible that postmenopausal women with lower levels of estrogen could exhibit lower expression of the TPH gene and protein or reduced activation of TPH, leading to reduced 5-HT synthesis and 5-HT levels in the CNS, potentially increasing the risk for mood disorders and eating disorders in postmenopausal women.

Despite the association between sex hormone levels and mood and eating disorders, not all postmenopausal women experience these conditions. A study has reported that sex hormone levels are not significantly associated with eating disorders in either premenopausal or perimenopausal women [135]. It is possible that the activity and expression of TPH and subsequent 5-HT synthesis, along with the activity of CNS serotonergic neurons, may adjust in response to dynamic changes in sex hormones during peri- and postmenopause in some women. These animal and human studies suggest a complex interaction between sex hormones and tryptophan metabolism, which may underly the sex differences in anxiety, depression, and eating disorders. Understanding sex differences and the effects of sex hormones on tryptophan metabolism pathways, especially in women, is critical for treating and preventing mental and eating disorders.

Sex differences in gut microbiota composition have been well documented, with men generally exhibiting higher levels of the Bacteroides and Prevotella groups than women [136]. Gene expression contributes to diet-induced sex-specific weight gain and fat distribution in both humans and laboratory animal models [137,138]. Dietary factors, rather than obesity itself, appear to induce sex-specific changes in gut microbiota composition. For instance, in rodent models, feeding mice an obesogenic HFD rapidly alters their gut microbiota at both the phylum and family levels, as well as the metabolite production of gut microbiota, often preceding the onset of diet-induced obesity [29]. In both sexes of mice, the consumption of an obesogenic HFD reduces the abundance of beneficial bacterial groups, such as Bifidobacteria [29], while increasing the abundance of Firmicutes, a microbiota phylum associated with enhanced energy harvest from food. These changes in turn promote obesity development and metabolic dysregulation in the host. Additionally, it has been reported that, using genetic mouse models with enhanced inflammation and diminished colonic barriers, Bifidobacteria play a crucial role in regulating the expression of tight junction protein, reducing the pro-inflammatory cytokines in mucus, and maintaining the epithelial barrier [139]. The findings of preclinical and clinical studies have indicated that a reduction in beneficial flora such as Bifidobacteria is closely linked with the development of chronic inflammation, a widely acknowledged contributing factor for various diseases, including cancer [140]. Interestingly, sex-specific differences in gut microbiota composition are observed in rodent models of HFD-induced obesity. The increase in Firmicutes in mice is substantially slower in females than in males [29].

Host gut microbiota also influence chromatin changes, such as DNA methylation and histone acetylation in colon epithelial cells. These modifications can alter the host epigenome, prime enhancers, and activate genes in the colon epithelium involved in functional signaling pathways related to metabolic dysfunction and cancer development. For example, the transplantation of microbiota from obese mice into germ-free mice has been shown to replicate HFD-associated epigenetic changes [29], leading to Dnmt1-independent DNA methylation changes in epithelial cells in Dnmt-deficient mouse models [141], modifications in the chromatin status of intestinal intraepithelial lymphocytes in germ-free mice [142], and changed host tissue chromatin features, resulting in alterations in host gene expression and physiology in germ-free mice [143]. These studies suggest that epigenetic changes may contribute to certain host outcomes related to the microbiome. Along the same line, germ-free animals reintroduced with microbiota from obese donors exhibit epigenetic changes at cancer-associated loci, indicating a potential connection between obesity and an elevated risk of colorectal cancer through these epigenetic modifications [144]. The interplay between epigenetic changes, inflammation, and tumorigenesis is likely influenced by the metabolism of obesogenic diets and gut microbiota, at least in laboratory mouse models.

Additionally, both the gut microbiome and the epigenomic responses, including chromatin characteristics, histone methylation, and histone acetylation in the colon epithelium, of mice fed an obesogenic diet are sex-specific [29]. A study by Lee et al. has reported that HFD feeding induces inflammation and cell proliferation in colon mucosa in both young (6 weeks old) and old (2 years old) rats of both sexes, while also causing gut dysbiosis characterized by decreased gut microbiota species richness and an increase in the abundance of specific microbial species, such as Clostridium lavalense that produce carcinogenic compounds, and an increase in the Firmicutes/Bacteroidetes ratio in old rats [145]. Notably, sex differences in gut microbiome alterations due to HFD feeding have been reported. For example, the abundance ratios of Akkermansia muciniphila and Desulfovibrio increase in response to HFD feeding in young rats of both sexes and old female rats, but not in old males [145]. Additionally, HFD feeding elevates the concentration of myeloperoxidase, an enzyme that produces reactive oxidants leading to tissue damage and inflammation, in the colon mucosa of male but not female rats [145]. These findings suggest sex differences in the composition of the gut microbiota in responding to obesogenic diets.

The consequences of the changes in microbiota associated with obesity include a reorganization of the microbiome, which subsequently affects disease risk. Thus, obesogenic diets and obesity exert differential effects on gut microbiota composition, abundance ratios, and microbial metabolite production in young and old males and females in laboratory mouse and rat models. This contributes to age- and sex-related differences in the development of inflammation and colon tumorigenesis, consistent with clinical studies that indicate a higher frequency of metabolic disorders and colon cancer in men compared to women [146,147,148,149,150,151].

Sex dimorphism has been identified as an important contributor to the distinct sex differences in the maturation and maintenance of the brain–gut–microbiome axis, contributing to differences in disease risk and resilience between males and females throughout their lifespans. The potential mechanisms underlying these sex differences in neural circuitry, which regulate feeding, metabolism, and mood, are only beginning to be understood and will continue to be explored. These mechanisms have largely been attributed to the neuroendocrine interactions in the brain–gut–microbiome axis involving sex hormones and neurotransmission, especially during developmental stages.

Steroid estrogens typically bind to and activate their cognate nuclear estrogen receptors (ERs) ERα and ERβ. Besides their well-known influences in developing and maintaining reproductive tissues and behaviors, estrogens also influence various non-reproductive targets, including gut microbiome and the CNS. For example, findings from preclinical and clinical studies suggest that estrogen and gut microbiota act together to regulate weight gain and lipid deposition [152], beneficially impacting on metabolic disorders such as obesity, diabetes, and cancer.

Lifestyle factors such as sedentary behavior, restricted mobility, and diets high in fat and sugar contribute to obesity development. Estrogens are essential for maintaining metabolic homeostasis in healthy premenopausal individuals. However, postmenopausal women, who experience decreased estrogen levels, often exhibit a reduced metabolic rate and become more susceptible to obesity development, fat deposition to the abdominal area, lipid disorder, cardiovascular diseases, and insulin resistance, all hallmark features of metabolic syndrome [137,153,154]. In humans, gut microbiota also play a significant role in the development and progression of various diseases, including non-alcoholic fatty liver disease [155] and colorectal cancer [156], suggesting a strong association between gut microbiota and risks for disease development and progression. Emerging evidence points to an interaction between estrogens and the microbiome that may enhance metabolic rate, lower body weight, and reduce adiposity, thereby decreasing the likelihood of metabolic diseases.

Importantly, genes for a variety of estrogen-metabolizing enzymes have been identified within gut microbiota in laboratory rodent models and humans [157], indicating that gut microbiota can metabolize dietary and endogenous estrogens and produce their metabolites that act as ER ligands. Among men, premenopausal women, and postmenopausal women, increased concentrations of estrogenic metabolites, along with reduced estrogen levels, positively correlate with increased microbial diversity, measured using fecal samples, and a reduced risk for breast cancer and metabolic diseases [158]. Thus, estrogenic metabolites may mediate host metabolism and alleviate symptoms associated with metabolic diseases and cancer. Although the underlying mechanisms remain incompletely understood, estrogen metabolism by the gut microbiota serves as a critical biomarker for evaluating host health and disease risk. The replacement of both estrogen and probiotics may synergistically improve gut microbiota composition and mitigate the chronic inflammation seen in metabolic diseases, potentially preventing fatty liver disease and cancer more effectively.

While the mechanisms by which 5-HT regulates energy metabolism, feeding behavior, and mood remain largely unclear, it is evident that studying sex differences in 5-HT neurotransmission is an area of active investigation, with results often inconclusive. Within the CNS, 5-HT is known to modulate mood, behavior, and cognitive functions. Low 5-HT levels are associated with depression, fatigue, and impaired cognition [168]. Serotonergic drugs, such as selective serotonin reuptake inhibitors (SSRIs), which interfere with the 5-HT transporter to increase the amount of 5-HT at the synaptic junctions among neurons, are commonly used to treat anxiety and depression by increasing synaptic 5-HT levels [169]. Furthermore, reduced levels of circulating tryptophan are linked to mood disorders, which can often be successfully treated with SSRIs [170].

Most 5-HT-expressing neurons project to the prefrontal cortex and hippocampus to integrate cognition and memory processes, to the hypothalamus to regulate energy homeostasis, and to the limbic system to mediate arousal and regulate mood. 5-HT exerts its effects in both the gut and the CNS through a variety of 5-HT receptors, which are divided into seven families (5-HT1 to 5-HT7). Except for the 5-HT3 receptor, which is a ligand-gated ion channel for Na⁺ and K⁺, the rest of the 5-HT receptors are G protein-coupled receptors [171]. While most 5-HT receptors are expressed in the CNS, 5-HT3 and 5-HT4 receptors are primarily found in the gastrointestinal tract. The key receptors involved in regulating anxiety include 5-HT1A, 5-HT2A, 5-HT2C, and 5-HT3. Notably, sex differences have been observed in the expression and function of these receptors, particularly in the context of depression and anxiety. In mammalians, including human and rodents, the serotonin transporter (SERT) plays a critical role in 5-HT signaling by moving 5-HT from the synaptic cleft into presynaptic neurons [172] where 5-HT is degraded. Thus, SERT reduces 5-HT content at the synaptic cleft and terminates 5-HT neurotransmission.

Estrogen influences 5-HT function through both genomic and non-genomic pathways, affecting the expression and activity of various serotonin receptors and SERT. In the CNS, the majority of 5-HT is synthesized in neurons located in the DRN of the midbrain. Both ERα and ERβ are expressed in serotoninergic neurons, particularly in the brain regions of the hypothalamus and DPN. ERα is expressed in the DRN of mice [132], while ERβ is expressed in the DRN of guinea pigs [133] and nonhuman primates [173,174]. Estrogen can regulate 5-HT neurotransmission through the activation of these receptors. For example, estradiol treatment has been shown to enhance 5-HT neuronal activity in the DRN of male and female rats [175,176]. Additionally, the selective activation of ERα using the agonist propylpyrazole-triol (PPT) increases 5-HT neuronal activity in the DRN of mice [132]. This activation is blocked when ERα is genetically deleted from the DRN’s serotonergic neurons [132], confirming that the effects of PPT on 5-HT neurons are dependent on ERα. Furthermore, the selective activation of ERβ tends to suppress, while activation of ERα has the opposite effect and induces, depression- and anxiety-like behaviors in rodent models [177,178,179].

In the gut, 5-HT involves hormonal and neuronal actions to regulate various critical physiological functions, including mucosal secretion via regulating releases of Cl⁻ and water into the lumen, nutrient absorption, vasodilation, gastrointestinal tract motor and sensory functions, as well as pain perception [41,99,214,215].

The neuronal action is highly conserved across various animal species including fish and mammals and can be detected by the cation current and potential related to depolarization and hyperpolarization, as well as by the expression of several neurotransmitters including substance P, acetylcholine, calcitonin gene-related peptide, and 5-HT [212,216]. In mammals, 5-HT in the ENS is released from EC cells, where it stimulates intrinsic afferent neurons that project to the mucosa, thus promoting the activation of enteric reflexes [217]. These reflexes help to regulate smooth muscle contractions and motility in the gastrointestinal tract.

The release of 5-HT is modulated by both chemical and mechanical stimuli in the mucosa, which are relayed through intrinsic afferent neurons to both descending and ascending interneurons, as well as to excitatory and inhibitory motor neurons that innervate and control the smooth muscle layers of the gut. 5-HT also acts as a neurotransmitter released from myenteric neurons involved in the peristaltic reflex neural pathway [217]. While these findings highlight 5-HT as a key mediator of gut motility, it worth noting that the depletion of endogenous 5-HT does not completely block peristalsis in the large intestine or impede gut transit in vertebrates [218].

When germ-free mice are colonized with microbiota from conventionally raised mice, observable changes occur in serotoninergic pathways, leading to increased gut motility. These changes are driven by 5-HT release from both EC cells and enteric neurons, and are associated with the proliferation of enteric neuron progenitors in the gut of adult mice [219]. This underscores the importance of 5-HT in ENS neurogenesis and neuroprotection [36,41,219].

Tryptamine, another tryptophan metabolite, may indirectly enhance gastrointestinal motility by triggering the release of 5-HT from the EC cells of guinea pigs [220]. It has been reported that, in in vitro mouse colonic epithelial cells, tryptamine directly affects ion secretion, food particle transition, and bacterial cell movement through the gut lumen [84]. This suggests that tryptamine plays a role in modulating both intestinal motility and gut microbiota interactions.

In addition to influencing the structure and function of the ENS through EC cells, the gut microbiota, along with its metabolites, also directly impacts intrinsic afferent neurons [43,221]. Research in germ-free mice has revealed reduced excitability in myenteric intrinsic afferent neurons, a condition that can be restored when the mice are introduced to a normal gut microbiota [222]. For example, Lactobacillus reuteri has been shown to increase neuronal excitability by enhancing the number of action potentials per depolarizing pulse, reducing calcium and potassium ion channel opening and decreasing slow after-hyperpolarization in intrinsic afferent neurons [43]. Specific bacterial strains also contribute to normal intestinal motor function. In germ-free rats, colonization with Lactobacillus acidophilus or Bifidobacterium bifidum can partially reverse neuromuscular dysfunction and delayed intestinal transit, while colonization with Escherichia coli or Micrococcus luteus can delay gut motility [223].

In summary, gut microbial metabolites, particularly tryptophan-derived compounds such as 5-HT and tryptamine play an essential role in regulating gut motility and energy metabolism. The microbiota influences the ENS through direct interactions with neurons and glial cells, and indirectly by modulating neurotransmitter release and neuronal excitability. Convincing evidence from the colonization of germ-free rodent models supports these results. These findings highlight the significant impact of the gut microbiome on gut function, neurogenesis, and overall energy homeostasis (Table 1).

In some cases, changes in neurotransmitter levels during stress may be attributed not to the stress itself, but to the gut microbiota. For example, Crumeyrollearias et al. have reported that in germ-free rats, concentrations of homovanillic acid (HVA) decrease in the anterior cortex, hippocampus, and striatum, leading to a reduced dopaminergic conversion rate and a lower HVA/dopamine ratio [228]. This change is unrelated to this open-field stressor, as the effects of this stressor show similar concentrations of norepinephrine, 5-HT, and its metabolite 5-hydroxyindole acetic acid (5-HIAA) in the anterior cortex and hippocampus, whether in germ-free or specific pathogen-free rats under stress. The reduced dopaminergic conversion rate in germ-free rats may result from a reduction in the expression or activity of DA degradation enzymes, such as catechol-O-methyltransferase and monoamine oxidase (MAO) [228]. Depression has been associated with a downregulated tryptophan metabolism, as evidenced by an increased plasma kynurenine/tryptophan ratio. Kelly et al. have demonstrated that germ-free rats administered microbiota from depressed patients also show an elevated HVA/dopamine ratio [229].

Animal studies have shown that the 5-HT neurotransmission system plays a crucial role in regulating feeding behavior [161]. The dysregulation of 5-HT neurotransmission not only affects cognition and memory but also disrupts energy metabolism and emotional responses related to feeding [162,163]. Enhanced 5-HT neurotransmission, as observed with drugs like SSRIs that block SERT and elevate synaptic 5-HT levels [169], generally suppresses eating in animals [200]. SSRIs also interact with other neurohormones that regulate feeding. For example, enhanced 5-HT neurotransmission through treatment with fluoxetine, an SSRI, reduces the expression of the orexigenic neuropeptide Y in the hypothalamus and suppresses feeding in rats [230]. The interaction between 5-HT and neuropeptide Y is species-dependent, as fluoxetine treatment simultaneously elevates the gene expression of both orexigenic neuropeptide Y and anorectic corticotropin-releasing factor 1 (CRF1) in the hypothalamus of female goldfish, leading to weight loss [231].

Interestingly, the suppression of eating due to enhanced 5-HT signaling through SERT inactivation by the SSRI fenfluramine is more pronounced in female rats than males, and more evident in the estrus than in the diestrus phase during the estrous cycle of female rats [232]. This suggests that sex differences in feeding suppression by 5-HT exist, potentially enhanced by estrogen. When estrous cycle phases are not considered, no sex differences in feeding responses to fluoxetine are observed [190]. It is noteworthy that the kinetics and metabolism of different SSRIs vary. For example, fluoxetine is metabolized more quickly in female mice than in males [233]. Additionally, it is possible that sex differences in the feeding response to SSRIs may be more apparent between estrous females and males if estrous cycles are considered.

Besides suppressing feeding and inducing satiation, the 5-HT system also regulates nutrient selection. For instance, in macronutrient selection tests in rats, enhanced 5-HT signaling through the administration of 5-HT [234,235], fluoxetine (an SSRI) [236], or 5-HT agonists [237], either systemically or via intra-hypothalamic administration, suppressed carbohydrate intake in male rats without affecting fat or protein consumption. In contrast to enhanced 5-HT signaling by 5-HT, SSRIs, or 5-HT agonists, 5-HT receptor antagonists enhance carbohydrate consumption and reduce carbohydrate satiation in rats [238]. These studies suggest that, in male rats, macronutrient selection regulated by 5-HT signaling is carbohydrate-specific, without affecting protein or fat consumption. In female rats, however, enhanced 5-HT signaling via the administration of the SSRI fluoxetine suppresses the consumption of fat and protein, but does not impact carbohydrate intake [239]. Thus, while macronutrient selection regulated by 5-HT signaling appears carbohydrate-specific in males, this is not the case in females.

Numerous studies have shown that gut microbiota have beneficial effects on behavior. For instance, Lactobacillus and Bifidobacterium species reduce anxiety-like and depression-like behaviors, alleviate mood disorders, and prevent stress-induced changes in colonic microbiota [240,241,242].

Early life is a critical period for the development of the CNS, including the hippocampus, and for the establishment of neurotransmission systems such as the serotonergic system. Clarke et al. have reported sex differences in the relationship between anxiety-like behavior and CNS 5-HT [70]. They have found that germ-free rats, particularly male rats, exhibit increased circulating tryptophan (a 5-HT precursor) and elevated hippocampal levels of 5-HT and a main 5-HT metabolite 5-HIAA, compared to conventionally colonized rats. In contrast, female germ-free rats do not show any changes in these parameters [70].

These findings suggest that gut microbiota regulate circulating tryptophan, influencing CNS 5-HT levels and 5-HT neurotransmission through a humoral route. The differences in CNS 5-HT levels between males and females may be influenced by the estrous cycle and sex hormones. The restoration of normal gut microbiota through transplantation or probiotic administration normalized circulating tryptophan levels and alleviated anxiety-like behavior in germ-free rats, but did not reverse CNS 5-HT levels [70]. The study suggests that gut microbiota regulates both CNS 5-HT neurotransmission and anxiety-like behavior, both of which are disturbed by the absence of normal gut microbiota. The resistant 5-HT levels indicate that CNS neurobiochemical and 5-HT neurotransmission changes caused by a lack of microbiota in early life are not easily reversed. Something during CNS development may have a stronger effect on CNS 5-HT neurotransmission than the normalized microbiota. These findings highlight the complexity of the relationship between CNS 5-HT and anxiety-like behavior, as restored microbiota do not fully reverse the biochemical changes in 5-HT neurotransmission caused by early-life microbiota disruption, and recovered anxiety-like behavior cannot be explained by still-elevated CNS 5-HT levels. We have reported that, in a rat study, while the changes in relative microbial abundance caused by adolescent chronic stress faded three weeks after the stress ended in young adults, changes in microbial metabolic profiles persisted into adulthood in male rats [243]. Therefore, metabolites associated with altered gut microbes may offer therapeutic potential for stress-related disorders.

5-HT is one of the most important monoamine neurotransmitters that regulates the development of the immature mammalian brain during the neonatal stages, and 5-HT neurotransmission continues to regulate anxiety throughout life. The effects of 5-HT on anxiety have also been studied in the prenatal context. Changes in endogenous 5-HT levels, influenced by MAO activity, can profoundly impact brain development and anxiety responses. In mice, sex differences in 5-HT deficits during the prenatal stage have been observed, with female mice showing reduced anxiety-like behavior in the elevated plus-maze and dark–light chamber tests, while male mice exhibit heightened anxiety-like behavior [244]. This underscores the differential effects of 5-HT on males and females, even during prenatal development. The study of sex differences in 5-HT-related anxiety could improve personalized treatment approaches for anxiety. A better understanding of how anxiety manifests in pregnant females or women of childbearing age would facilitate more effective treatments, thereby reducing its impact on developing infants.

Sex differences in stress responses are evident in studies of the 5-HT system at the hippocampus and midbrain. The cooperation between 5-HT and the HPA axis increases in response to stress, especially in females. In rats, females show increased binding at 5-HT1A receptors in regions such as the Cornu Ammonis (CA) 4 and CA1 of the hippocampus in response to stress, independent of estrous cycle phase [245]. Stress-exposed female rats also exhibit higher corticosterone levels, with a more pronounced decrease over time than males [245]. The administration of a 5-HT1A receptor antagonist increases neuronal activity in the midbrain DRN serotonergic neurons in both male and female rats, but decreases corticosterone levels only in male rats, not in female rats [246].

Both the HPA axis and central 5-HT neurotransmission, modulated by CRH, are necessary to modulate stress-induced adaptive coping behavior. The dysregulation of these systems increases susceptibility to affective disorders like depression and anxiety. Sex differences in the regulation of the HPA axis via 5-HT activity contribute to the higher incidence of stress-induced depression and anxiety in women compared to men. For example, in humans, men show a greater cortisol response to the 5-HT precursor 5-HTP than women, but in patients with affective disorders, this relationship is reversed, with women exhibiting heightened cortisol responses to 5-HTP [247]. Studies in genetically stress-sensitive mice (e.g., CRH receptor 2-deficient mice) further support the idea that increased 5-HT levels via SSRI administration can override dysfunctional HPA axis regulation and reduce anxiety [248]. These findings suggest that stress-induced sensitivity to 5-HT, particularly in females, may underlie their increased vulnerability to stress-related affective disorder.

Interestingly, SSRIs have distinct sex effects in humans, with women being less inhibited by SSRIs in 5-HT reuptake than men. Additionally, the effect of SSRIs in women depends on the different follicular, ovulatory, and luteal phases of the reproductive cycle (also known as the endometrial and menstrual cycles) [249,250], with greater 5-HT reuptake inhibition at the beginning of menstruation when estrogen and progesterone levels are low in women.

Therefore, the cooperation between the central regulation of the HPA axis and 5-HT neurotransmission is sex-dependent, with women having greater responsivity, which may contribute to the higher incidence of stress-induced depression and anxiety in women compared to men.