Utility of zebrafish-based models in understanding molecular mechanisms of neurotoxicity mediated by the gut–brain axis

Abstract

The gut microbes perform several beneficial functions which impact the periphery and central nervous systems of the host. Gut microbiota dysbiosis is acknowledged as a major contributor to the development of several neuropsychiatric and neurological disorders including bipolar disorder, depression, anxiety, Parkinson’s disease, Alzheimer’s disease, attention deficit hyperactivity disorder, and autism spectrum disorder. Thus, elucidation of how the gut microbiota–brain axis plays a role in health and disease conditions is a potential novel approach to prevent and treat brain disorders. The zebrafish (Danio rerio) is an invaluable vertebrate model that possesses conserved brain and intestinal features with those of humans, thus making zebrafish a valued model to investigate the interplay between the gut microbiota and host health. This chapter describes current findings on the utility of zebrafish in understanding molecular mechanisms of neurotoxicity mediated via the gut microbiota–brain axis. Specifically, it highlights the utility of zebrafish as a model organism for understanding how anthropogenic chemicals, pharmaceuticals and bacteria exposure affect animals and human health via the gut–brain axis.

1. Introduction

The gut microbiota is composed of symbiotic microorganisms (e.g., bacteria, viruses, archaea and fungi) that live in the intestines of vertebrates. The gut microbes are known for their numerous beneficial functions, namely fermentation of undigested carbohydrates, manufacturing of vitamins B and K, metabolism of drugs, bile acids and sterols, and protection against diseases (Clarke et al., 2014; Rowland et al., 2018). Interaction of the gut microbiota with the brain influences the host’s metabolism, immunity and health through its impacts on blood, immune cells, hormones, and neurons. Indeed, gut microbiota dysbiosis is currently acknowledged as a major player in the development of various neuropsychiatric and neurological disorders (Borrego-Ruiz and Borrego, 2023). The gut microbiota–brain axis reportedly promotes these diseases through several pathways linked to inflammation, endocrine disruption, neuronal injury and oxidative stress (Ortega et al., 2023; Zahedi et al., 2023a, b). Thus, the elucidation of how the gut microbiota–brain axis plays a role in health and disease conditions represents a potential novel approach to prevent and treat brain disorders (Tiwari et al., 2023).

The zebrafish (Danio rerio) is an invaluable vertebrate model that possesses conserved brain and intestinal features with those of humans (Kalueff et al., 2014; Flores et al., 2020). Based on the similarities with humans, zebrafish have been widely used as a powerful system to assess how different xenobiotics impact the gut–brain axis function (Li et al., 2022; Wang et al., 2022a, b, c; Huang et al., 2023). Moreover, neurochemical endpoints and behavioral evaluation using medium-to-high throughput screens in zebrafish are essential for understanding the molecular mechanisms associated with gut microbiota dysbiosis-associated neuropsychiatric and neurological disorders (Saleem and Kannan, 2018; Matsumoto et al., 2021). This chapter describes up-to-date findings on the utility of zebrafish in understanding molecular mechanisms of neurotoxicity mediated via the gut–brain axis. Specifically, it highlights the utility of zebrafish as a model organism for understanding how anthropogenic chemicals, pharmaceuticals and bacteria exposure affect animals and human health via the gut–brain axis. The possible mechanisms associated with psychobiotics in the treatment of neurological disorders associated with gut–brain disruption are also explored.

2. The gut microbiota–brain axis

The gut microbiota–brain axis represents the bi-directional communication between the central nervous system (CNS) and the digestive system which reportedly plays a major role in the health and disease (Carabotti et al., 2015; Ahmed et al., 2022; Mulder et al., 2023). The gut contains several trillions of microbes which are largely bacteria and some parasites, protozoa, yeasts, archaea helminths and viruses (De la Fuente, 2021; Uniyal et al., 2022). The gut microbiota is often considered an essential ‘organ’, which contributes to nutrient metabolism, maintenance of intestinal barrier homeostasis, immune defense, endocrine regulation, neurogenesis, and neurodevelopment (Guzzetta et al., 2022; Wasén et al., 2022). The gut microbiota composition is known to modulate CNS function via hormonal, neural, and immunological pathways. The vagus nerve is a direct communication link between the gut and the brain through which diverse gut-derived signals are transmitted, and has been associated with numerous immunological, gastrointestinal, and neurological disorders (Hashimoto, 2023; McVey Neufeld et al., 2023). Specifically, the gut microbiota sends signals to the brain via the vagus nerve, metabolic products and immune system, resulting in metabolic, developmental and physiological responses in the host (Chu et al., 2019; Komisaruk and Frangos, 2022). Thus, the functional interplay between the immune, endocrine and nervous systems underscores the need for proper functioning of the gut microbiome.

Previous investigations have shown that the gut microbiome influences brain function through modulation of stress, cognition and anxiety (Glover et al., 2021; Yun et al., 2021; Varanoske et al., 2022). Aberrant gut microbiome has been associated with pathology of neuroimmune-mediated disorders, cerebrovascular accidents, bipolar disorder, depression, anxiety, Parkinson’s disease, Alzheimer’s disease, attention deficit hyperactivity disorder, and autism spectrum disorder (Upadhyay et al., 2023; Zahedi et al., 2023a, b; Zhang et al., 2023; Liu et al., 2024). \Thus, the accurate understanding of this gut–brain axis link is indispensable for the development of novel therapeutic approach in the treatment of psychiatric conditions (Kamble and Dandekar, 2023; Zhao et al., 2023; Ferreiro et al., 2023). Unfortunately, the gut and its microbiota system are the first point of contact during dietary exposure to toxic environmental contaminants (Reygner et al., 2016; Gars et al., 2021; Parra-Martínez et al., 2022), and neurotoxicity can result from dysfunctional gut microbiota–brain axis (Ni et al., 2021; Wang et al., 2022a, b, c). It is noteworthy that psychobiotics (probiotic and prebiotics bacteria) reportedly enhance mood and cognitive functions in mice (Kim et al., 2021; Lee et al., 2023). Activities of psychobiotics in the gut modulate brain function by the regulation of neurotransmitters biosynthesis, production of active metabolites (e.g., short chain fatty acids), modulation of hormonal stress response through the hypothalamic-pituitary-adrenal (HPA)/hypothalamic–pituitary–interrenal axis, as well as the regulation of inflammatory mediators namely cytokines and chemokines (Chan et al., 2023; Feng et al., 2023). Thus, the delineation of the gut microbiota and the CNS responses to biological, physical or chemical exposures is an exciting emerging research area.

Although several investigations on the gut–brain axis have been carried out in rodents (Kim et al., 2021; Lee et al., 2023), the molecular mechanisms associated with the axis and neurotoxicity is not fully understood. Numerous studies have been performed recently in invertebrate systems (e.g., Caenorhabditis elegans and Drosophila melanogaster) to fully establish the contribution of this axis to neurotoxicity (Ortiz de Ora and Bess, 2021; Molina-Mateo et al., 2023). As an alternative vertebrate species, the zebrafish has been considered an established model organism in neurotoxicity research (Müller et al., 2017; Adedara et al., 2022; Lin et al., 2023a, b) to fully elucidate the biological functions of gut microbiome–brain axis due to its genetic tractability (Lin et al., 2023a, b; Ünal et al., 2023). The schematic diagram showing the bidirectional relationship between the gut microbiota and the brain is depicted in Fig. 1.

Fig. 1.

Schematic diagram showing the bidirectional relationship between the gut microbiota and the brain.

3. Neurotransmitter modulation by the gut microbiota

The brain performs several functions based on neurotransmission signals received from different neurons and glial cells (Shi et al., 2022). Brain homeostasis is largely dependent on excitatory neurotransmitters (e.g., glutamate, acetylcholine, and dopamine) and inhibitory neurotransmitters (e.g., gamma-aminobutyric acid (GABA), glycine, and serotonin). Evidence demonstrates that gut microbiota, through production of microbial metabolites and neurometabolites such as neurotransmitters, vitamins, and short chain fatty acids, impacts the brain function leading to behavioral modulation (Xie et al., 2022; Zhou et al., 2023). Gut microbiota metabolizes certain nutrients which are used by host cells to synthesize mammalian neuroactive products, namely GABA, monoamines (e.g., histamine, dopamine and serotonin) and tryptophan. Specifically, Lactobacillus spp., reportedly synthesizes acetylcholine whereas Bacillus spp., synthesize dopamine. Furthermore, Enterococcus spp., Candida spp., Streptococcus spp., and Escherichia spp. synthesize serotonin, Saccharomyces spp., Bacillus spp. and Escherichia spp., synthesize noradrenalin, and Bifidobacterium spp. and Lactobacillus spp. synthesize GABA (Stanaszek et al., 1977; Yunes et al., 2016; Scardaci et al., 2022; Ortega et al., 2023).

The metabolites act as neurotransmitter precursors or neurotransmitters, and travel through the systemic circulation and modulate brain functions. These neuroactive biomolecules activate nerve ganglia located in the submucosal and the myenteric plexuses of the enteric nervous system (ENS) (Spencer and Hu, 2020; Sharkey and Mawe, 2023). Thus, they act as indispensable mediators in the boundary between the gut and the brain. For instance, serotonin and glutamate from the enteroendocrine cells stimulate vagal afferents through transduction of luminal stimuli to the CNS (Li, 2007; Dockray, 2013). Moreover, over 90% of serotonin (5-hydroxytryptamine), which regulates peristalsis, mood, learning, memory, cognition, and reward predominantly resides in epithelial enterochromaffin cells of the gut (Zelkas et al., 2015; Bamalan et al., 2023). Dopamine is an essential neurotransmitter for motivation, cognition, reward, voluntary motor movements, pleasure, and satiety (Hamamah et al., 2022). It acts as a precursor for other norepinephrine and epinephrine (catecholamines). Norepinephrine is responsible for arousal and alertness in the waking state, detection of sensory signal, cognition, and working memory (Borodovitsyna et al., 2017; Sidorenko et al., 2023). Interestingly, more than 50% of dopamine in humans is synthesized in the gut, and peripheral dopamine levels can be regulated by gut microbiota (Hamamah et al., 2022). Indeed, activities of gut microbial genera Bacteroides, Bifidobacterium, Clostridium, Enterococcus, Lactobacillus, Prevotella, and Ruminococcus, via modulation of dopaminergic signaling, have been implicated in the pathogenesis of dopamine-related disorders (Hamamah et al., 2022).

Gut bacteria can regulate entero-endocrine signaling pathways via GABAergic neurotransmission, leading to several neurological disorders, including behavioral disorders and pain (Wong et al., 2003), ENS dysfunctions of intestinal motility, nociception, and gastric emptying (Hyland and Cryan, 2010). Lactobacillus, Lactococcus, and Streptococcus genera are acknowledged as safe or health-promoting microbes. Lactobacillus and Bifidobacterium strains isolated from the human intestine biosynthesize GABA (Barrett et al., 2012). Moreover, bacteria have also been reported to consume GABA. For instance, Evtepia gabavorous which solely depend on GABA-producing Bacteroides fragilis to grow has been described as a “GABA-eating” species (Strandwitz et al., 2019). Thus, it is well established that probiotic bacteria and gut bacteria are capable of producing glutamate or GABA (Barrett et al., 2012), and that GABA/Glutamate-producing gut bacteria modulate the host Glutamate/GABAergic systems (Bravo et al., 2011; Strandwitz et al., 2019). Moreover, the immune system is directly influenced by the gut microbiota, which consequently impacts the brain function. Indeed, the microbiota modulates the concentrations of corticosterone and corticotrophin-releasing hormone through the HPA axis (Hoban et al., 2016). The HPA axis plays a pivotal role in integrating peripheral gut functions with emotional and cognitive centers of the brain via mechanisms involving immune activation, enteric reflex, intestinal permeability, and entero-endocrine signaling. Thus, the interplay between the immune systems, ENS, and the HPA axis are indispensable in the maintenance of normal gut physiology and mental health (Hoban et al., 2016; Vodička et al., 2018; Dicks, 2022).

4. Zebrafish as an emerging model to investigate gut microbiota–brain axis

The zebrafish (D. rerio) is an established non-mammalian vertebrate model in neurotoxicity research owing to the small size, fast external development, high fecundity, optical transparency of larvae, convenient husbandry, as well as the genomic and physiological conservation (Miller et al., 2018; Canzian et al., 2022). Analogous to mammals, zebrafish have innate and adaptive immune defense systems (Novoa and Figueras, 2012; Levraud et al., 2022), the gut microbiota which helps in immune response, nutrient absorption and pathogen clampdown (Evariste et al., 2019). The fish gut has a large contact area with environmental pollutants, including microbes (Evariste et al., 2019). The relationship between host physiology and gut microbiota represents an exciting translational area of neurotoxicology. Zebrafish genome consists of about 70% genes in human, whereas almost 84% of genes associated with human disease are in zebrafish, thus highlighting its predictive value as a model organism to study the gut microbiota–brain axis (Gut et al., 2017; Wang et al., 2023). Zebrafish intestine is analogous to the mammalian intestine in development, structural composition, physiology, and biological function, thus making zebrafish a valued model to investigate the interplay between the gut microbiota and host health (Zhao and Pack, 2017; Andersen-Civil et al., 2023). Analogous to humans, the enterocytes, enteroendocrine cells, and goblet cells in matured epithelial cells in zebrafish intestines perform similar functions of nutrient absorption, hormone production, and mucus secretion, respectively (Flores et al., 2020). Moreover, zebrafish exhibits biochemical and behavioral responses comparable to rodents and humans (Canzian et al., 2022; Costa et al., 2023).

Myriads of zebrafish behaviors are essential in predictive neurotoxicology, and for the discovery and development of neurotherapeutic approaches (Costa et al., 2023; Gerlai, 2022). Neurobehavioral responses are the ultimate consequence of neuronal networking and signaling due to different stimuli from chemicals (chemotaxis), temperature gradient (thermotaxis), electric fields (electrotaxis), magnetic fields (magnetotaxis), light (scototaxis), and touch (thigmotaxis) (Rey et al., 2015; Sabadin et al., 2022). Thus, alterations in brain signaling and behavior of zebrafish due to microbiota dysbiosis can be effectively analyzed using established protocols. Indeed, the utility of zebrafish as a powerful model system to screen numerous antimicrobial drugs based on their putative CNS effects has been reviewed (Kotova et al., 2023). Several investigations in rodents have shown that gut biota dysfunction is associated with stress response, locomotion changes, anxiety-like behavior, depression-like behavior, nociceptive responses and social behaviors (Wang et al., 2022a, b, c; Olorocisimo et al., 2023; Huang et al., 2023). For instance, the novel tank diving test and light/dark tests are paradigms widely employed for measuring anxiety in fish. Indeed, scototaxis, thigmotaxis and freezing behavior, lower exploration rate, and increased time spent at the bottom of tank are predictive of anxiety-like responses (Sabadin et al., 2022). Thus, zebrafish is an invaluable model organism to investigate behavioral changes associated with gut microbial activities and psychobiotic activities with therapeutic effects.

5. Neurotoxic mechanisms of xenobiotics via the gut microbiota–brain axis in zebrafish

Exposure to environmental toxicants and toxins can adversely impact the gut micro-ecology leading to induction of gut microbiota dysbiosis, oxidative stress, inflammation, and enteric neurotransmission impairment (Roman et al., 2019). Alterations in gut health due to xenobiotic exposure can elicit neurobehavioral responses along with biochemical and histopathological changes in several extra-gastrointestinal tissues in exposed animals (Wang et al., 2022a, b, c; Aparna and Patri, 2023). Various neurotoxic mechanisms elicited by xenobiotics, mediated by the gut–brain axis in zebrafish, analyzed by a multi-end-point approach, are discussed in the current chapter. The substantial evidence from existing data highlights the utility of zebrafish in deepening our knowledge and understanding of the interactions between gut microbiota and the CNS. The cellular and molecular mechanisms of neurotoxicity associated with disrupted gut–brain axis in zebrafish are presented in Tables 1 and 2.

Table 1.

Neurotoxicity mechanisms associated with anthropogenic chemicals-mediated disruption of gut–brain axis in zebrafish.

Model	Experiment	Biochemical assay	Main results	References
Zebrafish larvae	Co-exposure of zebrafish larvae to lead (0.05 mg/L) and manganese (0.3 mg/L) for 7 days	Identification of 16S rRNA gene sequence. Expression of serotonergic and ABC transporter genes.	Reduced Proteobacteria proportion but increased Bacteroidetes and Firmicutes proportions. Up-regulation of serotonin signaling and metabolism genes (tph1b) as well as ATP-binding cassette G (i.e., abcg5 and abcg8)	Xia et al. (2023)
Adult zebrafish	Exposure of adult male zebrafish to 6:6 PFPiA (0, 1, 10 nM) for 28 days	Identification of 16S rRNA gene sequence	Increased Aeromonadaceae, Burkholderiaceae, Enterobacteriaceae Methylophilaceae and Xanthobacteraceae. Stimulation of inflammation, blood–brain barrier injury, and neuronal apoptosis	Zhang et al. (2023)
Adult zebrafish	Exposure of adult wild-type zebrafish to 1 mg/L imidacloprid under light phase extension (L:D=20:4 h; IMI-LL) and dark phase extension (L:D=4:20 h; IMI-DD) for 14 days	Identification of 16S rRNA gene sequence. Oxidative stress parameters. Expression of aralkylamine N-acetyltransferase and tryptophan hydroxylase genes	Decreased Proteobacteria abundance, but increased Fusobacteria proportion. Decreased SOD and CAT activities Increased MDA, ROS and 8-OHdG levels. Inhibited tryptophan metabolism pathway	Huang et al. (2023)
Zebrafish larvae/adult	Transient MPTP (1 μM) exposure at 48–96 hpf but raised without MPTP till 28 dpf	Identification of 16S rRNA gene sequence	Larval zebrafish: Increased Vibrionaceae but decreased Rhizobiaceae. Altered gut microbiome populations in adult male and female zebrafish	Dong et al. (2022)
Adult zebrafish	Continuous zebrafish exposure to Bisphenol F (2, 20, and 200 μg/L) from embryos (4 dpf) to adult stages (120 dpf)	Identification of 16S rRNA gene sequence, inflammatory genes and neurotransmitter metabolism	Decreased Bifidobacterium, Cetobacterium, Burkholderia-Caballeronia-Paraburkholderia and Halomonas, but increased Mycobacterium, Pseudomonas and Microbacterium proportion. Disruption in neurotransmitter metabolism	Wang et al. (2021)

Table 2.

Neurotoxicity mechanisms associated with pharmaceuticals- and bacteria-mediated disruption of gut–brain axis in zebrafish.

Model	Experiment	Biochemical assay	Main results	References
Adult zebrafish	Exposure of adult zebrafish to 6 and 60 μg/L enrofloxacin for 28 days	16S rRNA gene sequence. neuroinflammatory and neurotransmission systems	Elevated Bacteroidetes proportion, but reduced Firmicutes/Bacteroidetes ratio. Increased intestinal TNF-α, IL-6, GLP1 and 5-HT but decreased plasma cortisol and ACTH concentrations. Increased brain NPY, brain-derived neurotrophic factor and CRH	Tian et al. (2023)
Adult zebrafish	Exposure of adult zebrafish to cyclophosphamide at 0.05, 0.5, 5 and 50 μg/L for 2 months	16S rRNA gene sequence. Neurobehavior-related genes, dopamine-related genes, GABAergic- and serotonergic-related genes	Increased Fusobacteriales, Patescibateria, Rhodobacterals, Reyanellales and Staphylococcales proportions. Down-regulated neurobehavior-related genes (olig2 and lgals8), dopamine-related genes (th2 and drd2b) and GABAergic pathway (dlg4, grin2aa, grin2ab, opn3, opn5, tmtops, valopa and bdnf), whereas serotonergic-related genes (mao and tph1a) were up-regulated	Li et al. (2022)
Adult zebrafish	Exposure of zebrafish to flunitrazepam at 0, 0.2, and 5 μg/L for 30 days	16S rRNA gene sequence. ROCK, Rho A, and inflammatory genes expression	Increased Proteobacteria and Actinobacteria but decreased Fusobacteriota and Firmicutes populations. Intestinal expression of pro-inflammatory mediators, up-regulated ROCK and Rho A, down-regulated anti-inflammatory cytokine expression, Occludin and zonula occludens 1 proteins	Lin et al. (2023a, b)
Adult zebrafish	Exposure of zebrafish to morphine (40 mg/kg) and sinomenine (80 mg/kg) with or without antibiotic (vancomycin, 100 mg/L; gentamicin, 10 mg/L, and kanamycin, 5 mg/L)	Identification of 16S rRNA gene sequence. Expression of occludins, drd2a and htr2a genes	Morphine increased Fusobacteria but decreased Actinobacteria proportion leading to up-regulated Bacteroidetes/Firmicutes ratio. Downregulated occludin a and occludin b but up-regulated drd2a and htr2a gene expression	Chen et al. (2020)
Adult zebrafish	Exposure of zebrafish to morphine (40 mg/kg) and isorhynchophylline (100 mg/kg) with or without antibiotic (vancomycin, 100 mg/L; gentamicin, 10 mg/L, and kanamycin, 5 mg/L)	Expression of dopaminergic, serotonergic, glutamatergic and opioid system genes	Isorhynchophylline inhibited morphine-mediated down-regulation of Oprms and Oprds genes expression as well as the up-regulation of drd2a, htr2a, gad1 and gad2 genes expression	Chen et al. (2021)
Juvenile and adult zebrafish	Exposure of zebrafish to 250 μg/L triclosan from 30 dpf (juvenile zebrafish) to 90 dpf (adult zebrafish)	16S rRNA gene sequence. Biomarkers of inflammation, neurotransmission and apoptosis Histopathological examination	Increased Actinobacteria, Fusobacteria and Planctomycetacia proportion, with decreased Proteobacteria and Firmicutes. Elevated proinflammatory cytokines (IL6, IL1β, and IL21). Altered neurotransmission with gut and brain injuries	Wang et al. (2022a, b, c)
Adult zebrafish	Exposure of adult female zebrafish to L. casei, L. delbrueckii, and L. paracasei at a final concentration of 1.6 × 106 CFU/mL per aquarium and 200 mg/L of piracetam (a reference nootropic drug that reduces anxiety in humans) for 2 weeks	Identification of 16S rRNA gene sequence. Expression of glutamertagic genes	L. delbrueckii treatment increased Lactobacillus sp., Verrucomicrobium sp., Kaistia sp., Methylobacterium sp., Singulispira sp., Gordonia sp., and Mycobacterium sp. but decreased Legionella, Planctomyces, Flavobacterium, and Prevotella. L. delbrueckii treatment increased gad gene expression	Olorocisimo et al. (2023)
Adult zebrafish	Oral LGG supplementation at 106 CFUs/gram body weight to adult wild-type zebrafish chronically exposed to waterborne ethanol (0.01% v/v) for seven consecutive days	Histopathological examination of the zebrafish brain	Mitigation of ethanol-induced aberrant neurobehavior and brain injury in zebrafish	Aparna and Patri (2023)
Adult zebrafish	Exposure of adult wild-type zebrafish to Paraburkholderia sabiae (1.0 × 109 cell/L) in the rearing water twice a day at feeding times for 1 month	Identification of 16S rRNA gene sequence. GABAergic and serotogenic genes expression. Evaluation of neurotransmitter (taurine) concentration	Increased Rhizobiales populations namely Bradyrhizobiaceae, Pirellulaceae, Rhodospirillaceae and Xanthobacteraceae; decreased Actinomycetales populations namely Aeromonadaceae, Gordoniaceae, Gordonia, Nakamurellaceae, Nocardiaceae and Nocardia. Brain tph2 gene expression was increased	Ichikawa et al. (2023)

6. Anthropogenic chemicals and the gut microbiota–brain axis

Anthropogenic compounds (e.g., solvents, pesticides, and explosives) are extensively used in industry, agriculture, military operations, and medicine. Environmental contamination with metals can result from both natural and anthropogenic sources (Zhang et al., 2021a). The harmful effects, including neurotoxicity, associated with exposure to these anthropogenic contaminants in humans and wildlife are major global concern. Numerous studies have unequivocally validated the usefulness of zebrafish in unraveling the neurotoxic mechanisms of anthropogenic substances along the gut microbiota–brain axis (Zhang et al., 2021b; Grodzicki et al., 2021; Diao et al., 2021).

Manganese (Mn) is an essential dietary trace element, and the second most abundant metal occurring naturally in the earth’s crust, while lead is an environmental contaminant with neurotoxic effects (Erikson and Aschner, 2019; Botté et al., 2022). Environmental contamination with Mn and lead due to anthropogenic activities, namely inappropriate industrial discharge, combustion of leaded gasoline, and electronic waste recycling, may bring about the co-existence of these metals in real-life scenario (Guan et al., 2022). Previous investigation on the interactions between lead and Mn co-exposure, microbiota, and developmental neurotoxicity in zebrafish has been reported (Xia et al., 2023). Specifically, zebrafish larvae exposed to lead (0.05 mg/L) and Mn (0.3 mg/L), singly, and to the mixture of these metals for 7 days demonstrated that co-exposure induced lower locomotor activity and higher malformation rates than individual exposures. While individual metal exposure elicited no significant changes in gut microbial composition of zebrafish, combined exposure significantly reduced the Proteobacteria proportion, yet markedly elevated Bacteroidetes and Firmicutes. Unlike zebrafish exposed to Mn alone and lead alone, the genes associated with serotonin signaling and metabolism (e.g., tryptophan hydroxylase, tph1b) as well as ATP-binding cassette G (i.e., abcg5 and abcg8) were up-regulated in the combined exposure. These findings suggest the involvement of microbiota dysbiosis in neurotoxicity mediated by combined exposure to Mn and lead (Xia et al., 2023).

Perfluoroalkyl phosphonic acids and perfluoroalkyl phosphinic acids (PFPiAs) are important chemicals with various applications, namely surfactants in aerosols and antimicrobials, foam-dampening agents in textile industry, and repellents of grease, water and stain in cleaning formulations for rugs and carpets (Wang et al., 2016). Recent studies demonstrated the involvement of gut microbial dysfunction in the neurotoxicity associated with 6:6 PFPiA exposure in larval zebrafish (Zhang et al., 2023). Specifically, adult male zebrafish exposed to 0, 1, 10 nM of 6:6 PFPiA for 28 days demonstrated anxiety-like symptoms characterized by diminished frequency of entering the center and thigmotaxis, with increased velocity in the open field test. Notably, 6:6 PFPiA-exposed fish displayed significantly reduced exploration in the top during the novel tank test, whereas locomotion and scototaxis were markedly increased in the light/dark preference test. These findings highlight typical defensive behaviors and increased anxiety-like responses in exposed zebrafish. Gut microbiome data revealed that 6:6 PFPiA markedly increased the abundance of Gram-negative bacteria, such as Aeromonadaceae, Burkholderiaceae, Enterobacteriaceae Methylophilaceae, and Xanthobacteraceae, stimulating gut inflammation accompanied by blood-brain barrier injury, neuroinflammation, and neuronal apoptosis in the brain. Moreover, PFPiA exposure increased blood-brain barrier permeability, leading to its accumulation and activating binding to aryl hydrocarbon receptor in the fish brain (Zhang et al., 2023). Thus, chronic 6:6 PFPiA exposure may cause unpredicted neurotoxicity to human via mechanisms involving the gut microbiota–brain axis.

Imidacloprid, a neonicotinoid insecticide widely used in agricultural and residential settings, is known to elicit insecticidal activity and neurotoxicity in mammals via nicotinic acetylcholine receptors (Sheets et al., 2016; Boyd et al., 2020). The contribution of the gut microbiota–brain axis to the neurotoxicity associated with imidacloprid exposure in zebrafish has been reported (Huang et al., 2023). Specifically, exposure of adult wild-type zebrafish to 1 mg/L imidacloprid under light phase extension (L:D=20:4 h; IMI-LL) and dark phase extension group (L:D=4:20 h; IMI-DD) for 14 days demonstrated that imidacloprid exposure significantly decreased total superoxide dismutase and catalase activities, but increased malondialdehyde contents, reactive oxygen species (ROS), and 8-hydroxy-2 deoxyguanosine (8-OHdG) levels in the brain and gut of exposed zebrafish. The inhibition of antioxidant enzymes activities and the increase in oxidative stress biomarkers were aggravated in zebrafish exposed to imidacloprid and prolonged light. Microbiome analysis revealed that imidacloprid exposure markedly decreased the relative abundance of Proteobacteria, increased Fusobacteria proportion, and concomitantly inhibited tryptophan metabolism pathway. The imidacloprid-induced gut microbiota dysbiosis was also aggravated in zebrafish exposed to imidacloprid and prolonged light. Moreover, imidacloprid exposure markedly diminished melatonin and serotonin levels by inhibiting the transcriptions of genes encoding rate-limiting enzymes (aralkylamine N-acetyltransferase and tryptophan hydroxylase) in serotonin and melatonin synthesis. However, these biochemical and transcriptional status alterations were decreased in zebrafish exposed to imidacloprid and prolonged darkness (Huang et al., 2023). These findings highlight the contribution of gut microbiota dysbiosis to neurotoxicity associated with imidacloprid in zebrafish.

Zebrafish exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a well-known dopaminergic neurotoxin, at 48–96 h post-fertilization (hpf, sensitive developmental window) but raised in normal water without MPTP till 28 days post-fertilization (dpf) reportedly exhibited gut dysbiosis and male-biased neurobehavioral deficits (Dong et al., 2022). Specifically, transient MPTP exposure significantly increased Vibrionaceae but decreased Rhizobiaceae in larval zebrafish. In adult female fish, MPTP decreased the abundance of Beijerinckiaceae, Erysipelotrichaceae, Rhodobacteraceae, but increased Barnesiellaceae. Conversely, MPTP exposure decreased Aeromonadaceae, Chitinibacteraceae, Comamonadaceae and Shewanellaceae, but increased the abundance of Rhodobacteraceae in adult male zebrafish. Moreover, MPTP exposure caused locomotion deficits evidenced by hypoactivity in adult males under light conditions with reduced fast swim bouts. Social behavioral deficits were characterized by reduced time spent in area close to conspecific in the social preference test, as well as reduced transitions to the mirror area (Dong et al., 2022). These data demonstrated the involvement of altered gut microbiome composition on the persistent neurodevelopmental effect of MPTP exposure in zebrafish.

The extensive production and application of Bisphenol F, the main bisphenol A substitute, in the manufacturing of plastics for food, beverage, and other consumer products is known to cause environmental contamination and internal human exposure (Wang et al., 2021). Although Bisphenol F exposure has been associated with neurotoxicity, the underlying mechanisms involving gut microbiome dysbiosis and neurotransmitter metabolism disturbance was recently established (Gu et al., 2022). Continuous zebrafish exposure to sublethal Bisphenol F concentrations (2, 20, and 200 μg/L) from embryos (4 dpf) to adult stages (120 dpf) significantly decreased locomotor and social behaviors, but increased inflammatory genes expression in the brain, and compromised intestinal integrity. The 16S rRNA gene sequence data indicated that Bisphenol F treatment significantly diminished microbiota abundance and diversity in the exposed fish. Specifically, the relative abundance of Bifidobacterium, Cetobacterium, Burkholderia-Caballeronia-Paraburkholderia and Halomonas was reduced whereas Mycobacterium, Pseudomonas and Microbacterium were significantly increased following Bisphenol F exposure. Correlation analysis revealed a close relationship between gut microbiota dysbiosis and disruption in neurotransmitter-related metabolites namely glutamate, glutamine, leucine, proline, isoleucine, tyrosine and tryptophan (Wang et al., 2021). The vulnerable biochemical targets associated with disrupted gut–brain axis induced by anthropogenic chemicals in zebrafish are depicted in Fig. 2.

Fig. 2.

Vulnerable biochemical targets associated with disrupted gut–brain axis induced by anthropogenic chemicals in zebrafish.

7. Pharmaceuticals and the gut microbiota–brain axis

The extensive use of antibiotics in the treatment of microbial infections in aquaculture species, domestic animals, and humans is gradually becoming a major clinical concern due to their interference with gut microbiota–brain axis (Ibrahim et al., 2020). For instance, the anxiety-like behavior observed in zebrafish exposed to enrofloxacin has been associated with disrupted gut microbiota–brain axis (Tian et al., 2023). Adult zebrafish exposed to 6 and 60 μg/L for 28 days demonstrated that while enrofloxacin at low dose did not affect the behavior, high dose of enrofloxacin significantly increased the latency to enter the upper half and light area of the tanks, but decreased the durations in light area and average entry duration, thereby reflecting anxiety-like behavior. Moreover, enrofloxacin at 6 and 60 μg/L significantly elevated the abundance of Bacteroidetes, yet markedly reduced Firmicutes/Bacteroidetes ratio in the gut. Enrofloxacin significantly increased intestinal tumor necrosis factor-alpha (TNF-α), interleukin 6, glucagon-like peptide 1 and 5-hydroxytryptamine, but decreased plasma cortisol and adrenocorticotropic hormone concentrations. Moreover, enrofloxacin exposure increased the brain concentrations of neuropeptide Y, brain-derived neurotrophic factor and corticotropin-releasing hormone without affecting glial fibrillary acidic protein expression (Tian et al., 2023). The findings indicate the role of gut microbiota–brain axis disruption in anxiety-like behaviors of zebrafish chronically exposed to enrofloxacin. Thus, owing to antimicrobial and genetic similarities between zebrafish and humans, excessive exposure to antibiotics through gut microbiota–brain axis disturbance may impact neurological health.

Cyclophosphamide, a chemotherapeutic drug commonly used for the treatment of leukemia, ovarian cancer, lymphoma, multiple myeloma, breast cancer, sarcoma, and neuroblastoma, has been shown to induce neurotoxicity (Huehnchen et al., 2020; Ibrahim et al., 2023). Chronic cyclophosphamide exposure induces neurotoxicity in zebrafish by interfering with gut microbiota and brain neurotransmitters levels (Li et al., 2022). Specifically, cyclophosphamide exposure at 0.05, 0.5, 5 and 50 μg/L for 2 months significantly increased locomotion and anxiety, whereas cognition was markedly decreased in adult zebrafish. In contrast to high cyclophosphamide dose, the low dose (0.05 μg/L) of cyclophosphamide markedly down-regulated the expression of neurobehavior-related genes (olig2 and lgals8), dopamine-related genes (th2 and drd2b), and GABAergic pathway (dlg4, grin2aa, grin2ab, opn3, opn5, tmtops valopa and bdnf), whereas serotonergic related genes (mao and tph1a) were up-regulated. Furthermore, cyclophosphamide increased the relative abundance of Fusobacteriales, Patescibateria, Rhodobacterals, Reyanellales and Staphylococcales in the intestine of exposed fish (Li et al., 2022). These findings indicate that cyclophosphamide, through modulation of gut microbiota–brain axis, elicit locomotion, cognition, and anxiety-related changes in zebrafish.

Flunitrazepam is a benzodiazepine commonly prescribed for insomnia treatment and used as a hypnotic or preoperative sedative in medicine (Doyno and White, 2021). The abuse of benzodiazepines including flunitrazepam is common among drug addicts (Dåderman and Edman, 2001) and rapists during sexual assault (Busardò et al., 2019). Experimental evidence from zebrafish chronically exposed to flunitrazepam at 0, 0.2, and 5 μg/L for 30 days indicate that flunitrazepam-induced neurotoxicity was associated with gut microbiota–brain axis disruption (Lin et al., 2023a, b) Specifically, flunitrazepam exposure significantly increased the intestinal abundance of Proteobacteria and Actinobacteria but decreased Fusobacteriota and Firmicutes population, thereby causing pathological injury in exposed fish. Moreover, intestinal expression of pro-inflammatory mediators namely interferon-gamma, interleukin-1beta and TNF-α were up-regulated, whereas anti-inflammatory cytokine IL-10 expression was down-regulated. While flunitrazepam significantly downregulated tight junction proteins, Occludin and zonula occludens 1, it markedly increased intestinal permeability, Rho-associated kinase (ROCK) and Ras homolog gene family, member A (RhoA) expression in the exposed fish. Flunitrazepam-mediated increase in the brain lipopolysaccharide and malondialdehyde levels was accompanied by pathological changes and disruption of nucleotide and amino acid metabolic pathways in the brain. Flunitrazepam-induced neurotoxicity via the gut microbiota–brain axis was corroborated by correlation analysis, thus unraveling a possible molecular neurotoxic mechanisms associated with psychoactive drug abuse.

The clinical application of morphine as an efficient analgesic is reportedly limited due to tolerance and addiction effects (Badshah et al., 2023). To simulate human drug-dependent behavior commonly found among drug addicts, non-human animals are usually subjected to conditioning place preference (CPP) test. Earlier investigations demonstrated that sinomenine, an alkaloid with potent pharmacological actions, effectively inhibited acquisition of place preference in morphine-dependent mice (Fang et al., 2017). However, to elucidate the functional mechanism between morphine dependence and intestinal microbiome, a CPP test was performed following antibiotic administration to morphine-dependent adult zebrafish (Chen et al., 2020). Specifically, findings from zebrafish injected with morphine (40 mg/kg) and sinomenine (80 mg/kg) with or without antibiotic (vancomycin, 100 mg/L; gentamicin, 10 mg/L, and kanamycin, 5 mg/L) revealed that morphine-induced CPP was accompanied by significant increase in Fusobacteria, yet decreased Actinobacteria proportion leading to up-regulated Bacteroidetes/Firmicutes ratio in the gut microbiota. Moreover, morphine significantly down-regulated occludin a and occludin b, but up-regulated dopamine receptor D2 (drd2a) and 5-hydroxytryptamine receptor 2A (htr2a) gene expression in the brain and intestine of treated zebrafish. While sinomenine administration efficiently inhibited morphine-induced CPP in the absence of antibiotic, it failed to suppress morphine dependence after antibiotic treatment, thus evidencing the role of gut microbiota–brain axis in morphine-dependent zebrafish (Chen et al., 2020). These findings highlight the possible significance of antibiotic-mediated disruption of intestinal microbiota in the therapeutic efficacy of sinomenine on morphine addiction.

Moreover, the therapeutic effect of isorhynchophylline, an indole alkaloid from Uncaria rhynchophylla, on morphine dependence has been demonstrated to involve the gut microbiota–brain axis (Chen et al., 2021). Specifically, zebrafish injected with morphine (40 mg/kg) and isorhynchophylline (100 mg/kg) with or without antibiotic (vancomycin, 100 mg/L; gentamicin, 10 mg/L, and kanamycin, 5 mg/L) revealed that isorhynchophylline administration effectively inhibited morphine-induced CPP and dysbacteriosis without antibiotic pretreatment. However, it was ineffective in suppressing morphine dependence following antibiotic treatment. Similar to rodents, morphine-treated zebrafish exhibited marked down-regulation of mu (Oprm) and delta opioid receptors (Oprd) which are acknowledged to mediate opioid dependence (Chen et al., 2021). Isorhynchophylline inhibited morphine mediated down-regulation of Oprms and Oprds gene expression as well as the up-regulation of drd2a, htr2a and glutamate decarboxylase 1 (gad1) and gad2 gene expression in the brain and intestine of treated zebrafish. However, following antibiotic-driven microbiota dysbiosis, isorhynchophylline failed to ameliorate morphine-induced behavior and biochemical alterations, thus demonstrating the relationship between gut microbiome, opioid receptors and morphine addiction (Chen et al., 2021). These findings highlight the role of the gut microbiota–brain axis, and provide new neurotherapeutic approach to drug addiction.

Triclosan is a broad-spectrum antibacterial widely used in industries and in the production of consumer products namely cosmetics, body washes, soaps and toothpastes (Alfhili and Lee, 2019). Human exposure to triclosan may occur through ingestion of contaminated water and food products. Excessive exposure to triclosan has been associated with several human health problems including allergic reactions (Weatherly and Gosse, 2017) and neurotoxicity in hippocampal neurons cell line HT22 (Liu et al., 2021) and animals (Ruszkiewicz et al., 2017; Pullaguri et al., 2023). Previous investigation into the role of intestinal flora disorder on neurotoxicity associated with triclosan exposure indicated that zebrafish exposed to 250 μg/L from 30 to 90 dpf showed suppressed swimming behavior associated with autism- and depression-like behaviors in exposed fish (Wang et al., 2022a, b, c). Triclosan-induced gut microbiota dysbiosis was characterized by marked increase in Actinobacteria, Fusobacteria and Planctomycetacia proportion, decrease in Proteobacteria and Firmicutes, and elevated proinflammatory cytokines (il6, il1β, and il21) in exposed fish. Moreover, triclosan-exposed fish exhibited marked inhibition of acetylcholinesterase activity and dopamine level but increased neuronal apoptosis and histopathological injuries in the brain and the gut. However, dietary supplementation with short-chain fatty acids (SCFA, 5% in feed), which regulates intestinal microbiota and sympathetic nerve activation, effectively assuaged the noxious effects elicited by triclosan exposure (Wang et al., 2022a, b, c), suggesting the involvement of the gut–brain axis in triclosan-induced neurotoxicity. The vulnerable biochemical targets associated with disrupted gut–brain axis induced by pharmaceuticals in zebrafish are depicted in Fig. 3.

Fig. 3.

Susceptible biochemical targets accompanying gut–brain axis disruption by pharmaceuticals in zebrafish.

8. Bacteria exposure and the gut microbiota–brain axis

Psychobiotics are beneficial bacteria which have shown efficacy in the treatment of mental health disorders via interaction of the gut–brain communication networks (Sarkar et al., 2016). Psychobiotics elicit antidepressant- and anxiolytic-like effects characterized by alterations in neural, cognitive and emotional parameters (Foster and McVey Neufeld, 2013; Du et al., 2020). Psychobiotics were originally called probiotics because ingestion of an appropriate amount exerts positive psychiatric influence on psychopathology (Cheng et al., 2019). Gram-positive bacteria, namely Bifidobacterium and Lactobacillus families, which do not elicit immunological reactions due to lack of pro-inflammatory lipopolysaccharide chains are commonly exploited as probiotics (Sarkar et al., 2016). Previous studies have demonstrated that some psychobiotics effectively reduce anxiety- and stress-related behaviors as well as improve cognition and mood via the gut microbiota–brain axis (Cheng et al., 2019; Misra and Mohanty, 2019). Importantly, the elucidation of exact mechanism of action associated with known psychobiotics is increasingly gaining the attention of researchers.

The potential psychobiotic activities of Lactobacillacea family comprising of Lacticaseibacillus paracasei, Lacticaseibacillus casei and Lactobacillus delbrueckii have been described previously (Olorocisimo et al., 2023). Although anxiety is an adaptive response to dangers through which an organism decides to fight or flee (Meacham and Bergstrom, 2016), maladaptive anxiety refers to persistent and excessive fear of threats in the absence of a stressor. Earlier investigations on the psychobiotic activity have demonstrated that L. delbrueckii reduced anxiety-like behavior in zebrafish via the gut microbiota–brain axis in zebrafish (Olorocisimo et al., 2023). Adult female zebrafish exposed to L. casei, L. delbrueckii, and L. paracasei at a final concentration of 1.6 × 10⁶ CFU/mL per aquarium and 200 mg/L of piracetam (i.e., a reference nootropic drug that reduces anxiety in humans) for 2 weeks demonstrated that L. delbrueckii and piracetam significantly decreased anxiety-like behavior in treated zebrafish. Specifically, L. delbrueckii increased exploration rate and the time spent in the upper half, but decreased the latency to enter the upper half area in the novel tank test. Moreover, L. delbrueckii treatment altered zebrafish gut microbime by increasing the abundance of Lactobacillus sp., Verrucomicrobium sp., Kaistia sp., Methylobacterium sp., Singulispira sp., Gordonia sp., and Mycobacterium sp., but decreased Legionella, Planctomyces, Flavobacterium, and Prevotella. Treatment with L. delbrueckii increased glutamic acid decarboxylase (gad) gene expression in zebrafish gut and brain (Olorocisimo et al., 2023). These findings highlight that L. delbrueckii, but not L. paracasei and L. casei, exhibited anxiolytic-like effects by modulating gut microbiota and increasing gad1 expression in gut and brain of zebrafish, supporting a therapeutic potential as a psychobiotic in pathological states of anxiety.

Lactobacillus rhamnosus Gorbach Goldin (LGG) is a human gut bacterium well-acknowledged to be a standard health index for flourishing gut–brain axis (Shulman et al., 2022; Zhou et al., 2022). The beneficial effect of LGG supplementation on chronic ethanol-induced neurobehavioral response of zebrafish has been reported (Aparna and Patri, 2023). Specifically, oral LGG supplementation at 10⁶ CFUs/gram body weight to adult wild-type zebrafish chronically exposed to waterborne ethanol (0.01% v/v) for seven consecutive days demonstrated that LGG mitigated ethanol-induced alteration in habitat preference, locomotor activity and cognitive dysfunction. Histopathological data demonstrated that LGG ameliorated ethanol-induced intestinal epithelial disruption and neuronal pyknosis in periventricular gray zone of zebrafish brain. These findings demonstrate that probiotic LGG supplementation mitigate ethanol-induced aberrant neurobehavior and brain injury in zebrafish (Aparna and Patri, 2023). Thus, LGG supplementation may be a novel therapeutic approach for neurological disorders associated with chronic ethanol ingestion in human health.

Adult wild-type zebrafish administered Paraburkholderia sabiae (1.0 × 10⁹ cell/L) in the rearing water twice a day at feeding times for 1 month markedly reduced anxiety-like behavior in the treated zebrafish. Specifically, P. sabiae-exposed zebrafish exhibited increased average acceleration, average speed, and total distance. P. sabiae treatment increased Rhizobiales populations, whereas Actinomycetales populations were reduced in the fish gut. Functional analysis demonstrated that P. sabiae treatment increased brain taurine concentration whereas the expression of GABAergic genes, namely gabra1 and gad1 remained unaffected. While brain tph2 gene expression was increased, other serotonergic genes namely htr1aa, tph1a, tph1b, mao-z, slc4a6a, and slc4a6b as well as the brain serotonin concentrations were unaffected in treated zebrafish. These findings demonstrate that P. sabiae decreases anxiety-like behavior in adult zebrafish via modulation of the gut microbiome–brain axis. The vulnerable biochemical targets associated with disrupted gut–brain axis induced by bacteria exposure in zebrafish are depicted in Fig. 4.

Fig. 4.

Vulnerable biochemical targets associated with disrupted gut–brain axis due to bacteria exposure in zebrafish.

9. Conclusion

Overall, based on existing data, the utility of zebrafish in unraveling the gut microbiota–brain axis in zebrafish is well-established. Similar to mammals, microbial symbiosis in zebrafish is known to regulate both the central and peripheral nervous system functions by modulating gene expression, neurotransmitters, brain signaling, and aberrant behaviors. Zebrafish sensitivity to numerous neurotoxic anthropogenic chemicals, pharmaceuticals, and biological agents is exciting because of the similarities between the genome and intestinal environments of zebrafish and humans. Thus, zebrafish represent a valid, alternative, complementary, and predictive vertebrate model to screen, develop and elucidate the mechanisms of potential therapeutic agents for microbiota-associated neuropsychiatric diseases.