Early life interaction between the microbiota and the enteric nervous system

Abstract

Recent studies on humans and their key experimental model, the mouse, have begun to uncover the importance of gastrointestinal (GI) microbiota and enteric nervous system (ENS) interactions during developmental windows spanning from conception to adolescence. Disruptions in GI microbiota and ENS during these windows by environmental factors, particularly antibiotic exposure, have been linked to increased susceptibility of the host to several diseases. Mouse models have provided new insights to potential signaling factors between the microbiota and ENS. We review very recent work on maturation of GI microbiota and ENS during three key developmental windows: embryogenesis, early postnatal, and postweaning periods. We discuss advances in understanding of interactions between the two systems and highlight research avenues for future studies.

INTRODUCTION

Recent studies have begun to uncover fundamental aspects of the interactions between the gastrointestinal (GI) microbiota and its host: how early life microbiota affects the maturation of the little brain, also known as the enteric nervous system (ENS). The microbiota that reside in the GI tract form a complex ecosystem comprising bacteria, archaea, viruses, and fungi (51). The colon harbors the largest microbial population (10¹⁴ bacterial cells) in the human body. Within the GI tract, luminal microbiota communicate with the closely juxtaposed ENS embedded in the gut wall, which contains complex neuronal circuits that govern vital gut functions. Several studies have established the ENS as a major target of microbiota signals that impact host physiology and behavior (13, 16, 20, 32, 33, 35, 39, 45, 46, 53, 62, 70). However, when interactions between microbiota and the ENS begin and whether there are health implications associated with their disruption during critical developmental windows are new research frontiers.

Here, we present evidence for early life interactions between bacterial microbes and the ENS from conception to adolescence and discuss potential signaling mechanisms between the two systems. We also consider the impact of early life antibiotics on gut function, emphasizing the importance of proper development of microbiota and ENS interactions, and highlight research avenues for future studies.

THE MOUSE AS A MODEL FOR MICROBIOTA-ENS INTERACTIONS

Mice are pivotal for providing mechanistic insights and study of the physiological significance of microbiota-ENS interactions. Almost 99% of mouse genes are shared with humans, and the GI microbiota of mice and humans have core similarities that include a general predominance of bacterial phyla Firmicutes and Bacteroidetes confined to similar regional compartments (22, 37). Mice are useful as model systems due to the vast library of commercially available transgenic animals, the relative ease of custom-made genetic models using contemporary methods, and the ability to apply gnotobiotics to manipulate host-microbiota interactions in germ-free animals by inoculating them with specific or a consortium of microbes (37). We will discuss development of microbiota and ENS in humans where possible, but our focus is on recent advances that further understanding of microbiota-ENS interactions in mouse models.

THE ENS AND GI MICROBIOTA DEVELOP CONCURRENTLY

The mouse is the best-studied mammal for the development of the ENS. Research on the mouse GI tract has enabled creation of a detailed timeline of key milestones that demonstrate the concurrent maturation of the ENS and microbiota and hence development of putative interactions during three critical developmental windows: embryogenesis, early postnatal, and postweaning periods (Fig. 1).

Fig. 1.

Developmental timeline for the mouse gut. Schematic diagram depicting development of enteric nervous system and microbiota during 3 critical windows: embryogenesis (E0.5–E18.5), early postnatal (P0–P10) period, postweaning period (P2–P49), and adulthood. Key developmental milestones are flagged at various time points along the time line. ENCCs, enteric neural crest cells; GI, gastrointestinal; nAChRs, nicotinic acetylcholine receptors.

Embryogenesis

Substantial development of the ENS occurs during embryogenesis. There have been decades of concerted research efforts in advancing our understanding of ENS development during embryogenesis. In mice, the ENS is mainly derived from cells of the vagal neural crest, with smaller contributions from truncal and sacral levels, and Schwann cell precursors. During embryogenesis, a complex array of molecular and cellular mechanisms orchestrates migration of neural crest cells to and within the developing GI tract, proliferation of these precursors, and differentiation into enteric neuronal and glial subtypes (27, 50). More recently, enteric neurons from human and mouse embryos were found to express specific chemicals/transmitters, receptors, and ion channels and are already electrically active (26, 27, 29, 44). Additionally, embryonic enteric neurons have begun to form functional connections with each other via neurotransmitters including acetylcholine and 5-hydroxytryptamine (5-HT), contributing to survival and differentiation of later born enteric neurons (26, 27).

There has been intense debate for over 100 years about whether the fetal environment is sterile or whether microbiota are transferred from mother to fetus in utero (59). The vast majority of past research established the sterile womb paradigm. However, a renaissance in the hypothesis that the fetus encounters bacteria before birth has come from studies applying modern techniques and demonstrating the presence of bacteria in the placenta, amniotic fluid, and umbilical cords in normal pregnancies. Furthermore, the composition of gut microbiota varies with length of gestation, and the first stool of infants (meconium) that was also previously believed to be sterile has been found to contain bacteria, albeit in low abundance (51, 59). This debate continues today because next-generation sequencing applied to these studies does not differentiate live versus dead microbes, and environmental contamination is a major potential confounder in low biomass tissues. Of note, a recent heroic study examined samples from 537 women and found no bacteria in healthy placentas (19). Although this study seemed to put an end to the longstanding debate, it was immediately contested by a study identifying bacteria-like morphology and sparse bacterial signals in human fetal meconium at midgestation (60) and another showing the presence of bacterial DNA in human and mouse fetuses during mid- and late-gestation and identifying viable bacteria from murine fetal tissues (73). Therefore, this issue remains contentious, and more studies are warranted.

Early Postnatal Period

Although many properties of the GI tract develop by 24 wk gestation in humans so that it can process milk feedings by term, the function of the GI tract is not mature at birth (28). Mice are born with immature GI tracts relative to humans; newborn mouse gut has similar maturity to that of preterm (26 wk) infants, whereas postnatal day (P)10 gut from mice is similar to full-term infant gut. Research on the early postnatal development of the murine ENS is an emerging field, arising only in the last 15 years (23). The motility patterns of the early postnatal mouse colon are still immature, and enteric neurons in its main underlying circuitry within the myenteric plexus are still acquiring their neurochemistry and maturing in their morphology and electrophysiological properties (23). Less is known about the development of the other division of the ENS, the submucous plexus, but we do know that submucosal neurons differentiate later than myenteric neurons (23, 41).

To be vaginally delivered full-term and exclusively breastfed constitutes the gold standard for a healthy infant microbiota. Human and mouse infants acquire much of their founding microbiota during the early postnatal period. The early colonizers are critical for the establishment of later microbes in a process known as microbial succession (28, 37). At birth, the intestines of infants are rapidly colonized by bacteria from their immediate environment, either from the mother’s vagina or skin depending on mode of delivery. In humans, initial colonizers are generally facultative anaerobes, belonging to the Enterobacter, Enterococcus, Staphylococcus, and Streptococcus genera, due to the anoxic environment in the intestine during and shortly after birth. As these facultative bacteria flourish, the resulting lowered redox potential allows obligate anaerobic bacteria, such as Bifidobacterium, Bacteroides, and Clostridium to proliferate and become the predominant genera associated with early life (28). Similarly, in mice, an initial bloom of Streptococcus occurs after birth and is replaced by Lactobacillus after P3 (56). Lactobacillus ferments milk lactose and casein and is another microbe associated with vaginally delivered human babies that is found in breast milk (28). Whether babies are fed breast milk or formula influences the development of microbiota. Notably, breast milk components, particularly glycoproteins and human milk oligosaccharides (HMOs), selectively enhance the growth of Bifidobacterium, the dominant bacteria in breast-fed infant gut (58). In contrast, infants given bovine milk-based formulas devoid of HMOs have a lower abundance of Bifidobacterium (58).

Very low birth weight (VLBW), preterm (born before 37 wk of pregnancy), and sick infants often experience rapid vaginal or Cesarean section deliveries and spend a significant amount of time after birth in the Neonatal Intensive Care Unit (NICU). Although this is pivotal for survival of critically ill babies and minimizes damage to brain development, some common practices in the NICU can increase risk of infection in the infant and disturb the GI microbiota. For instance, respiratory support may introduce oxygen leading to abnormal survival of aerobic bacteria immediately after birth. Most babies in the NICU are exposed to antibiotics and receive bovine milk formulas and/or milk from preterm mothers that have a different mix of HMOs compared with full-term breast milk, all of which impacts microbial succession and potentially affects development and disease susceptibility later in life (28). Understanding the impact of environmental insults immediately after birth on the microbiota, ENS, and their interactions can improve the health outcomes of preterm, vulnerable babies and should be the agenda for future research.

Postweaning Period

The postweaning period is defined as the period during and immediately after weaning. Mice have more accelerated early life compared with humans, and adolescence or puberty can begin in mice as early as postnatal day (P) 18–28, when juvenile mice are weaned from the dam (22). The vital components of the ENS would have been acquired during embryogenesis and early postnatal life; thus, postweaning development is likely to involve continued formation of synapses in the enteric network that have commenced in earlier stages and fine tuning of the circuit connections that underpin maturation of GI functions. Indeed, the electrophysiological properties and synaptic profile of enteric neurons are still immature at P10 (24). Research on the postweaning development of the ENS has emerged only within the last 3–5 years. We recently demonstrated that between pre- and postweaning periods, there is a significant increase in the somata size of enteric neurons and numbers of synaptophysin+ appositions. The architecture of the enteric plexi is still maturing as the ganglia containing neurons and glia are stretched further apart. Although there is no appreciable change in neurochemistry of myenteric neurons, substantial maturation in submucosal neurochemistry still occurs during the postweaning period (57). Schwann cell-derived enteric neurons and S100β+ glia that are found in the intestinal mucosa are all still developing postweaning (35, 67). Moreover, enteric neurogenesis could persist even into adulthood (38).

In humans, major shifts in microbiota are triggered by the transition from mother’s milk to solid food during weaning at around 4 mo of age. A mature microbiome appears to be established by 3 yr of age in healthy full-term babies and remains stable through childhood into adolescence and adulthood (37). Accordingly, we found that significant increases in abundance and communities of fecal microbiota occurred during the postweaning period in mice (33). The introduction of solids increases substrate diversity for intestinal bacteria, such that the fecal microbiota begins to resemble that of their mother’s (56). The increased diversity of gut microbiota established during this period would produce new pathways of fermentation, thereby influencing their interaction with the ENS, an area for future research.

IMPLICATIONS OF ANTIBIOTIC EXPOSURE DURING CRITICAL DEVELOPMENTAL WINDOWS

A healthy gut microbiota consists of coexisting pathogenic (pathobionts) and symbiotic bacteria. Disrupting this balance alters host susceptibility to disease. Antibiotics were developed and used as medicines around the 1940s; shortly thereafter, they earned the title of “miracle cure” by effectively treating infections, thereby leading to a dramatic decrease in death rates and serious illnesses. Fast track to modern times, epidemiology shows that babies and young children receive the highest antibiotic exposures globally (2) (Table 1). Although antibiotics are essential in many circumstances, recent startling findings show that early life exposure to antibiotics has long-lasting detrimental impacts on health, including increased susceptibility to several diseases such as GI and mental disorders, obesity, metabolic dysfunction, and allergies later in life (18, 49). Healthy human pregnancies are accompanied by dramatic changes in gut microbiota composition, with increased abundance of Proteobacteria and Actinobacteria from the first to the third trimester (51). Several antibiotics such as β-lactams and macrolides are considered safe for use during pregnancy and account for 80% of all medications prescribed to pregnant women (40). Recently, maternal exposure to antibiotics, even before pregnancy, has been associated with increased risk of childhood hospitalized infections, including gastroenteritis (47). Moreover, maternal antibiotics are linked to childhood obesity (40). However, the impact of maternal antibiotics during pregnancy on the fetus, especially its ENS, is understudied. VLBW and preterm infants are particularly vulnerable to bacterial infections, and thus antibiotic treatment is a common practice in the NICU (28), but antibiotics do not distinguish between symbionts and pathobionts. Exposure to antibiotics during critical developmental windows disrupts microbial succession and may allow pathobionts to gain a foothold early in life, thereby causing poor health.

Table 1.

Mouse studies demonstrating importance of microbiota on the ENS

Method	Age Treated/Age Examined	Effects	Reference
Abolishment of GI microbiota
Broad-spectrum antibiotic cocktail (ampicillin, metronidazole, vancomycin, neomycin ± amphotericin-B) in drinking water or by oral gavage for 2–5 wk	Adult (8–12 wk old)	Reduced mucosal S100β+ glia in the ileum	(33)
	Treated: at P0, P21, or P42. Examined: adult	Decreased 5-HT levels in the colon	(67)
	Adult female mice	Slower GI transit	(20)
	3-wk-old male mice	Disrupted myenteric neurons and glia, upregulation of BDNF signaling in the ileum	(6, 13)
	Treated: 10-day-old pregnant mice until weaning of pups, or newly weaned pups until P36. Examined: male mice	Temporal modulation of nitrergic neurons, delayed whole gut transit in TashTTg/Tg model of Hirschsprung but increased transit in WT	(63)
Germ-free	Adult (8 wk old)	Disrupted mucosal S100β+ glia in the ileum	(33)
	Adult	Decreased 5-HT levels in the colon and perturbed GI motility	(60, 67)
	Adult (8–12 wk old)	Slower intestinal transit and had immature ENS	(20)
	Adult	Affected intrinsic sensory neuron electrophysiology and expression of calbindin	(43, 44)
	P3	Disrupted myenteric plexus of jejunum and ileum, decreased muscle contractions	(16)
Microbial Conventionalization
Germ-free mice with SPF microbiota	Treated: at P0, P21, or P42. Examined: P56 adult	Restoration of 5-HT levels	(67)
Germ-free/antibiotic-treated mice with indigenous spore-forming microbes	Treated: at P42. Examined: P56 adult	Restoration of 5-HT levels and motility	(67)
Humanized mice were generated by oral gavage germ-free mice with fecal microbiota from two healthy human donors	Treated: at 4–6 wk of age or at 8 wk old. Examined: adult	Increased Tph1 expression and 5-HT concentrations	(60)
Transfer bedding from SPF cages into germ-free cages for 4 wk	Adult	Restored intrinsic sensory neuron electrophysiology and expression of calbindin	(43, 44)
Initial oral gavage of germ-free mice with fecal/cecum content from age-matched mice then co-housed	Treated: from 4 wk old. Examined: adult (8 wk old)	Network of glial cells in the lamina propria restored	(33)
	Treated: adult (for 15 days). Examined: adult	Induced maturation of ENS, restored ENS via 5-HT4-dependent pathway	(20)
Depletion of GI microbiota
Single daily dose of oral vancomycin	Treated: from P0–P10. Examined at P10/11	Disrupted colonic ENS, 5-HT, and motility	(31)
Vancomycin in drinking water for 3 wk	Treated: from day of weaning. Examined at P42–49	Disrupted myenteric and submucosal neurons and colonic motility	(32)
Ampicillin in drinking water for 2 wk	Adult (8–16 wk old)	Prolonged whole gut transit, decreased enteric neuron density and proportion of nitrergic neurons	(68)

5-HT, 5-hydroxytryptamine; BDNF, brain-derived neurotrophic factor; ENS, enteric nervous system; GI, gastrointestinal; P, postnatal day; SPF, specific pathogen free; Tph1, tryptophan hydroxylase 1; WT, wild-type.

Combating infectious diseases with antibiotics is one of the greatest medical revolutions, but the new discoveries of potentially negative health consequences imposed on our next generation are a conundrum, especially because little is known about antibiotic-induced disease mechanisms in the infant GI tract. Although the rule is that antibiotics should be used when “the benefits outweigh the risks,” research should focus on identifying measures that reduce risks and inform early life antibiotic stewardship, as this remains one of the most difficult challenges for the clinical practice.

EFFECTS OF MICROBIOTA ON THE DEVELOPING ENS

Although the ENS and microbiota develop concurrently within the GI tract, the crosstalk between the two systems and how the microbiota affect development of the ENS remain unclear. The vast majority of researchers have focused on interactions of microbiota and ENS in adult mice that are either germ free or have their gut microbiota effectively abolished by a cocktail of broad-spectrum antibiotics for a lengthy period of time (Table 1). Conventionalizing these mice by colonizing their guts by exposure to fecal material of normal specific pathogen free mice or specific microbes partially reverses effects of the (pseudo)germ-free state on the ENS (Table 1). Studies examining postweaning development have used the same methods to show that the microbiota regulate gut neuromuscular function (13) and the influx of enteric glial cells into the mucosa of the small intestine (35). More recently, we and others opted for a model that depletes, but does not abolish, gut microbiota (32, 33, 71). We exposed mice to a single antibiotic, vancomycin, during the postweaning period and showed that significant shifts in microbiota community composition (dysbiosis) are associated with disruptions to the neurochemistry and function of myenteric neurons and, importantly, the poorly studied submucosal neurons, leading to impaired colonic motility in young adult mice (33).

To our knowledge, there have been no studies of the impact of microbiota on the developing ENS during embryogenesis and extremely few studies on the early postnatal period (Table 1). The ENS in the small intestine of germ-free mice is abnormal at P3 (16). Whether disruption to the developing ENS is due to the lack of microbiota is unclear, as other systems are significantly perturbed in germ-free mice (72). Our recent work shows that oral administration of vancomycin to neonatal mice daily from birth to P10 disrupts their colonic microbiota, motility, myenteric neurochemistry, and activity (32). Although this supports the view that microbiota are important in regulating ENS development, the possibility that vancomycin has toxic effects directly on the ENS cannot be excluded (21). Furthermore, vancomycin’s effects on the microbiota and ENS differed between early postnatal exposure and postweaning exposure, and its impact appears to be greater when administered to younger animals (32, 33). These data emphasize the importance of studying roles of microbiota and impact of antibiotics at different stages in life.

MECHANISMS BY WHICH MICROBIOTA SIGNAL TO THE ENS

Currently, there is little understanding of the mechanisms by which microbiota influences ENS development. However, several signaling mechanisms, mainly discovered in the mature ENS, are worth investigating for involvement during development.

Toll-Like Receptors

The expression of pattern recognition receptors (toll-like receptors, TLRs) by the ENS, immune cells, and intestinal epithelial cells allows these cells to sense gut microbes directly. In particular, TLR2 and TLR4 have modulatory roles on enteric neurons and glia and neuromuscular function (10, 12, 13). TLR2 can regulate the release of neurotropic factors from smooth muscle cells (10). A cell wall component of gram-negative bacteria, lipopolysaccharide (LPS), can activate TLR4 and phosphorylation of JNK-1 in synergy with the free-fatty acid, palmitate, in mice given a high-fat or western diet to induce loss of nitrergic enteric neurons and dysmotility (3, 61). Very recently, nestin+ enteric neuronal precursor cells in adult mouse colon were reported to express TLR2 and TLR4 (71). Exposure to ampicillin impaired neurogenesis of nitrergic neurons, an effect prevented by supplementation with the TLR2 agonist, lopoteichoic acid (LTA) (71). In contrast, LTA has been reported to induce loss of nitrergic myenteric neurons in cultured duodenal muscularis preparations (52). These discrepancies could be due to TLR2 having different roles along the GI tract or an effect induced by examining preparations in culture.

Microvesicles

Microvesicles, typically 20–400 nm in diameter, are constantly formed and shed by most microbes and contain lipids, phospholipids, LPS, DNA, and RNA. Recent studies have supported the concept that microvesicles are a major mode of communication between gram-positive bacteria, particularly of the Lactobacillus species, and the ENS (1, 68). Microvesicles isolated from Lactobacillus rhamnosus JB-1 and DSM in culture recapitulate effects of the parent bacteria on gut motility (1, 68). Similar to eukaryotic microvesicles, it is possible and indeed likely that these vesicles contain microbial-derived small inhibitory RNAs that can impact host cell development and function.

Transcription and Neurotrophic Factors

A recent study reported that gut microbiota signal to enteric neurons in the colon via the transcription factor aryl hydrocarbon receptor (AHR) to regulate motility (53). Brain-derived neurotrophic factor and its high affinity receptor tropomyosin-related kinase B are affected in the ENS of juvenile mice treated with antibiotics (6).

Microbial Metabolites

Symbiotic bacteria metabolize foods that are indigestible by the host, thereby harvesting energy, nutrients, and metabolites for the host’s benefit. Notably, short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate are derived from fermenting indigestible carbohydrates and proteins. It was hypothesized that the developing fetus is exposed to maternally derived SCFAs via the placenta. SCFAs may regulate ENS development by increasing the growth rate of human neural progenitor cells via influencing expression of neurogenesis, proliferation, and apoptosis-related genes (69). Acetate and butyrate can induce tryptophan hydroxylase 1 (TPH1) expression in human-derived enterochrommaffin cell models (BON cells) (62). Moreover, very recently it was demonstrated that SCFA-producing gut microbiota can modulate gut-extrinsic neuron activity, which relay information to brainstem sensory nuclei and efferent sympathetic premotor neurons to regulate GI transit (48). Besides SCFAs, other metabolites including α-tocopherol, cholate, deoxycholate, p-aminobenzoate, and tyramine elevate 5-HT and increase TPH1 expression in RIN14B chromaffin cell cultures (70). Moreover, a longer, 5-carbon branched-chain fatty acid, isovaleric acid, produced by bacterial fermentation of leucine acts directly on colonic smooth muscle to induce relaxation (7).

Enteroendocrine Cells and Immune System

The GI tract is also a major contributor to the immune and endocrine systems, which probably develop concurrently with the microbiota and the ENS during critical developmental periods but are even less understood. The possibility that immune cells, especially muscularis macrophages, act as mediator signals between the microbiota and ENS during development has been reviewed elsewhere (55); we will focus on the contribution of enteroendocrine cells interspersed within the intestinal mucosa.

Besides the traditional paracrine action involving diffusion of mediators, enteroendocrine cells including peptide YY (PYY) secreting L cells, and serotonin (5-HT) secreting enterochromaffin cells can communicate to the ENS by synaptic transmission (4, 8). L cells, which also contain glucagon-like peptide-1 (GLP-1), are found throughout the small intestine, but in mice, their highest density is in the colon. Unlike the L cells in the small intestine whose activity is meal (glucose) dependent, colonic L cells express bile acid receptors and free fatty acid receptors 2 and 3 for SCFAs strategically to respond to bacterial metabolism (17). Indole, a metabolite of tryptophan produced by microbes, modulates GLP-1 secretion from L cells, which can then activate GLP-1 receptors expressed on enteric neurons (11, 15). Several studies of the adult ENS and our work on neonatal exposure to vancomycin have shown that microbes signal to the ENS by modifying 5-HT metabolism of enterochromaffin cells and its signaling from the mucosa (20, 32, 43, 62, 70). De Vadder and colleagues (20) further showed that microbiota modulate enteric neurogenesis by activating 5-HT₄ receptors. In contrast, exposure to vancomycin just postweaning did not involve 5-HT metabolism in the mucosa (33). Thus, more studies investigating how the microbes signal to the developing ENS via 5-HT or other processes are warranted.

THE MOUSE AS A PRECLINICAL MODEL REVEALING THE IMPORTANCE OF MICROBIOTA-ENS INTERACTIONS IN DISEASE

In the best-studied human congenital GI disorder, Hirschsprung disease (HSCR), the absence of enteric ganglia in the distal GI tract results from abnormal ENS development due to disorders in multiple genes. Mice with haploinsufficiency in many HSCR genes recapitulate the HSCR phenotypic aganglionic bowels (9). Mouse Hirschsprung models have an altered microbiome community structure, and it was recently suggested that there may be a common microbiome signature for Hirschsprung (66). Microbiota may contribute to the severity of HSCR disease, as antibiotic treatment delayed the development of megacolon around weaning but brought forward the onset of severe constipation later in life due to temporal modulation of the proportion of nitrergic neurons in the transition zone of the TashT^Tg/Tg Hirschsprung model (66). Interestingly, whereas most studies demonstrate the importance of microbiota on the ENS, a study of a Sox10 mutant Hirschsprung zebrafish model found that transplantation of the intestine with ENS precursors produced a normal-like ENS along the length of the intestine and corrected inflammation induced by dysbiosis (64). This suggests that the ENS can modulate intestinal microbiota to maintain gut health, which warrants further investigation.

Microbiota and ENS are each involved in several pathologies that begin early in life, but effects of their interaction have not been examined in most cases, especially since the ENS is typically the underappreciated system under investigation. Mouse models of neurodevelopmental disorders such as Autism spectrum disorder have altered microbiota community compositions, and their GI and behavioral abnormalities can be modulated by microbiota (31), but effects on the developing ENS are not yet known. Additionally, there is sexual dimorphism in the gut microbiota and susceptibility to stress-induced psychiatric disorders (30, 34). A recent study in mice administered accumulative insults in utero and during childhood showed that there were sex-specific differences in behavioral phenotypes, gene expression in the medial prefrontal cortex, and gut microbiota during adulthood (63). However, gender differences in maturation of gut microbiota and the ENS in normal and diseased states remain largely unexplored and should be pursued in future research. In Parkinson’s disease, GI disorders often precede motor dysfunction in patients. LPS can modulate aggregation of α-synuclein (5), a presynaptic protein expressed in the nervous system in the brain, gut epithelial cells, and the ENS (42, 65). Furthermore, the ENS is now recognized to be a therapeutic target for treatment of metabolic disorders (36). Dysbiosis in diet-induced diabetic mice causes neuropathy of nitrergic myenteric neurons (25, 52). Thus, elucidating targets within the enteric circuitry and pathways between microbiota and ENS signaling is a new avenue for research. It is also important not to assume that dysbiosis is inevitably the bearer of bad news, since adaptation of GI microbiota communities may represent compensatory responses to host distress signals.

CONCLUSIONS AND FUTURE DIRECTIONS

The GI tract is an organ where multiple systems coexist, and it is not a coincidence that the microbiota and ENS develop concurrently. There are numerous challenges in the field, including consideration of the complexities of the microbiota ecosystem, ENS and their intermediaries within the gut, sexual dimorphism, understanding the interaction of microbiota and ENS across the lifespan, and design of preclinical models that mimic the human condition. Future studies should aim to provide mechanistic insights into the crosstalk between the microbiota and ENS during various developmental windows. Antibiotics are important drugs, especially for their life-saving qualities. Identifying the short-term and lasting impacts of antibiotic usage during critical developmental windows on the host would advance antibiotic therapy by revealing preventative measures against its unwanted side effects and improve the health and wellbeing of our next generation.

GRANTS

This research was supported by National Health and Medical Research Council of Australia Project Grants APP1099016 to J. P. P. Foong, J. C. Bornstein, and T. C. Savidge and P30-DK56338, PO1-AI152999, U01-AI24290, and R01-AI10091401 to T. C Savidge, as well as the University of Melbourne International Research and Fee Remission Scholarship to L. Y. Hung.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.P.P.F. prepared figures; J.P.P.F. drafted manuscript; J.P.P.F., L.Y.H., S.P., T.C.S., and J.C.B. edited and revised manuscript; J.P.P.F., L.Y.H., S.P., T.C.S., and J.C.B. approved final version of manuscript.