Other Sections Abstract Introduction Development of Enteric Neurons in the Gastrointestinal Tract Potential Therapy for Enteric Nervous System Deficient Diseases Differentiation of Enteric Neurons From Mouse Embryonic Stem Cells The 5-hydroxytryptamine Receptors Acute Actions of 5-hydroxytryptamine 4 Receptor Agonists in the Gut In Vivo Enteric Neurogenic Actions of Brain-derived Neurotrophic Factor in the Distal Gut Enteric Neurogenic Actions of the 5-hydroxytryptamine 4 Receptor Agonist in the Distal Gut Enteric Neurogenic Actions of the 5-hydroxytryptamine 4 Receptor Agonist in the Mouse Proximal Gut Effects of Selective Serotonin Reuptake Inhibitors on Neurogenesis Figure References

We have reported a novel approach in vivo to reconstruct the enteric neural circuitry that mediates a fundamental distal gut reflex.1-4 The neural circuit insult was performed in guinea pigs by rectal transection and subsequent end-to-end one layer anastomosis.5,6 Brain-derived neurotrophic factor (BDNF) applied locally at the anastomosis promoted regeneration of the distal gut reflex pathways in the enteric nervous system (ENS) and repaired the anal dysfunction.6,7

BDNF, however, has serious detrimental actions such as pro-inflammatory action on the anastomosis and thus we explored a small molecule candidate promoting enteric neurogenesis. We found a brief report showing that some 5-hydroxytryptamine 4 receptor (5-HT 4 R) agonists increased neuronal numbers and length of neurites in enteric neurons in vitro from immunoselected neural crest (NC)-derived precursors in an abstract form.8

Therefore, we subsequently explored a novel approach in vivo to reconstruct the enteric neural circuitry in the distal gut of guinea pigs9 and rats10 by application of an 5-HT 4 R agonist.

Jackie D Wood introduced our studies and Gershon's work as follows.11 Laboratories of Takaki at Nara Medical University, Japan and Gershon at Columbia University, New York have convincing evidence for enteric neurogenesis from indwelling stem cell populations.6,7,9,12 Takaki directed her attention to restoration of functional recto-anal inhibitory reflex connections and defecation following resection and anastomosis in a guinea pig model and published the first evidence that serotonergic action at the 5-HT 4 R subtype might be a signal for differentiation of ENS stem cells into functional neurons that become "wired" into a reflex circuit that restored functional large intestinal motility.6,7,9

The idea that the gut contains serotonergic neurons has been controversial. Because enteric neurons take up 5-hydroxytryptamine (5-HT),13-17 5-HT-containing neurons have been called "5-HT accumulating," to suggest that their 5-HT is derived from the enetrochromaffin (EC) cell.18 Mucosal 5-HT, however, does not normally reach myenteric ganglia.19 Nevertheless, stimulated enteric neurons release 5-HT;19 moreover, 5-HT receptor blocking anti-idiotypic antibodies20 and acute 5-HT depletion21 abolish putatively serotonergic slow excitatory post-synaptic potentials (EPSPs) mediated by 5-HT 1p .22,23 Criteria needed to establish 5-HT as an enteric neurotransmitter24 were finally completed by the discovery that enteric neurons express tryptophan hydroxylase 2 (TPH2).25

The gut contains a large 5-HT pool in EC cells and a smaller 5-HT pool in the ENS. During development, enteric neurons are generated asynchronously. Serotonergic neurons, which arise early, affect development/survival of later-born dopaminergic, gamma-aminobutyric acidergic, nitrergic, and calcitonin gene-related peptide-expressing neurons and are essential for gastrointestinal motility. The 5-HT biosynthesis depends on tryptophan hydroxylase 1 (TPH1) in EC cells and on TPH2 in neurons; therefore, deletion of TPH2, but not TPH1, decreased myenteric neuronal density and proportions of dopaminergic and gamma-aminobutyric acidergic neurons. Addition of 5-HT to cultures of isolated enteric neural crest-derived cells (ENCDCs) promoted total and dopaminergic neuronal development. Moreover, serotonergic neurons promote development/survival of some classes of late-born enteric neurons, including dopaminergic neurons.26

Serotonin (5-HT) might be an enteric neuronal growth factor which was first suggested by observations that 5-HT 2B receptors are neuronal, developmentally regulated, and promote development of neurons from isolated ENCDCs.27 Subsequently, 5-HT 4 Rs were demonstrated to be neuroprotective and to promote the development of new neurons from adult stem cells.12,28 Because enteric neurons are born asynchronously29 and serotonergic neurons are among the first to arise,29-31 it follows that serotonergic neurons coexist with dividing ENCDCs, some of which to receive synapses.32 It is thus plausible that serotonergic input influences the fate of ENCDCs.

We applied a 5-HT 4 R agonist, mosapride citrate (MOS) locally at the anastomosis in guinea pigs9 or orally by the drinking water in rats10 for 2-4 weeks. Furthermore, we examined neurofilament (NF)-, 5-HT 4 R- and 5-bromo-2'-deoxyuridine (BrdU)-positive cells in the newly formed granulation tissue at the anastomotic site 2-4 weeks after enteric nerve circuit insult. Possible neural stem cell markers, anti-distal less homeobox 2 (DLX2)- and p75 neurotrophin receptor (NTR)-positive cells, and NF-positive cells were also examined during the same time period.

We also developed a spontaneously contracting gut differentiated from mouse embryonic stem (ES) cells (ES gut) using an embryoid body (EB)33 culture system.34 Although in these ES guts, enteric neural networks were not identified, we succeeded in forming dense enteric neural networks by MOS added only during EB formation.35

In the small intestine in H-line: Thy1 promoter green fluorescent protein (GFP) mice, we obtained clear 3-dimensional in vivo imaging of enteric neurons that were newly generated through oral application of MOS after gut transection and anastomosis.36 BrdU- and NF-positive cells were identified as new enteric neurons.