Significance
The gut microbiota affects several physiological processes, including gut motility. Here we observed that germ-free mice have an immature enteric nervous system (ENS) that is normalized upon colonization with a normal microbiota. We identified the mechanism of communication between the microbiota and enteric neurons as the initiation of serotonin release and subsequent activation of the 5-HT4 receptor. This demonstrates a strong interaction between the microbiota and the ENS and indicates potential mechanisms linking microbial dysbiosis to gastrointestinal disorders. The ability to modulate the microbiota, e.g., by diet, will open new perspectives of research in neurogastroenterology.
Abstract
The enteric nervous system (ENS) is crucial for essential gastrointestinal physiologic functions such as motility, fluid secretion, and blood flow. The gut is colonized by trillions of bacteria that regulate host production of several signaling molecules including serotonin (5-HT) and other hormones and neurotransmitters. Approximately 90% of 5-HT originates from the intestine, and activation of the 5-HT4 receptor in the ENS has been linked to adult neurogenesis and neuroprotection. Here, we tested the hypothesis that the gut microbiota could induce maturation of the adult ENS through release of 5-HT and activation of 5-HT4 receptors. Colonization of germ-free mice with a microbiota from conventionally raised mice modified the neuroanatomy of the ENS and increased intestinal transit rates, which was associated with neuronal and mucosal 5-HT production and the proliferation of enteric neuronal progenitors in the adult intestine. Pharmacological modulation of the 5-HT4 receptor, as well as depletion of endogenous 5-HT, identified a mechanistic link between the gut microbiota and maturation of the adult ENS through the release of 5-HT and activation of the 5-HT4 receptor. Taken together, these findings show that the microbiota modulates the anatomy of the adult ENS in a 5-HT–dependent fashion with concomitant changes in intestinal transit.
The gastrointestinal tract is unique in the body, as it has an intrinsic nervous system, the enteric nervous system (ENS), composed of the outer myenteric plexus and the inner submucosal plexus. The ENS has several essential physiologic functions, such as regulation of gastrointestinal motility, fluid secretion and absorption, and blood flow (
1). In rodents, most enteric neurons are formed during embryogenesis and early postnatal life (
2⇓⇓–
5). During this process, a small subpopulation of Sox10-expressing neural crest-derived cells colonizes the foregut, subsequently undergoes proliferation, and then colonizes the entire bowel, giving rise to both neurons and glial cells (
6⇓–
8). Furthermore, enteric neuronal stem cells, which express markers such as Nestin, have been reported in the postnatal murine intestine (
9⇓–
11). While it was long thought that no enteric neurons were formed after postnatal day 21 in mice, except in cases of inflammation or injury (
2⇓–
4), a recent study has demonstrated that the ENS undergoes continuous renewal in adult mice, with apoptosis balancing neurogenesis (
12). Thus, this report demonstrates that the adult ENS is capable of maturation and plasticity, but the mechanisms driving these processes remain unknown.
The microbiota colonizes the gastrointestinal tract after birth with continuous maturation during the first year of life (
13). Concomitantly with colonization of the gastrointestinal tract and maturation of the mucosal immune system, the ENS undergoes extensive development during the early postnatal life (
14). Accordingly, germ-free (GF) mice, which lack a gut microbiota, have early postnatal structural and functional abnormalities of the ENS (
15). These features can be reversed by colonization with the microbiota from a conventionally raised (CONV-R) donor (
16).
About 90% of the body’s serotonin (5-hydroxytryptamine, 5-HT) is produced by enterochromaffin (EC) cells, a type of enteroendocrine cells which are present in the epithelium of the gut (
17). Recently, two studies have reported that the gut microbiota is able to induce mucosal 5-HT secretion in the gut (
18,
19). The concept that mucosal and neuronal 5-HT are distinct pools is supported by the fact that different forms of the rate-limiting synthetic enzyme tryptophan hydroxylase (TPH) are used by neuronal and nonneuronal cells, with TPH2 used by neurons and TPH1 used by EC cells (
20). While mucosal 5-HT is strongly proinflammatory (
21,
22), activation of the 5-HT4 receptor (5-HT4R) in the ENS exerts neurogenerative and neuroprotective actions (
4,
23).
In this study, we tested the hypothesis that the gut microbiota modulates the function and the anatomy of the ENS through release of 5-HT and activation of the 5-HT4 receptor (5-HT4R). Colonization of GF mice reduced intestinal transit time and was associated with the release of 5-HT. We studied GF and colonized
Tph1-deficient mice and demonstrated that mucosal 5-HT is neuroprotective in the early phases of colonization. Next, we studied the respective roles of mucosal and neuronal 5-HT in the observed phenotype through inhibition of TPH1 and TPH2 with parachlorophenylalanine (PCPA) or depletion of neuronal 5-HT with reserpine. Finally, using pharmacological modulation of the 5-HT4 receptor, we established a link between the gut microbiota and neuronal activation of 5-HT4Rs.
