Background  It was recently reported that some 5‐HT₄‐receptor agonists increased neuronal numbers and length of neurites in enteric neurons developing in vitro from immunoselected neural crest‐derived precursors. We aimed to explore a novel approach in vivo to reconstruct the enteric neural circuitry that mediates a fundamental distal gut reflex.

Methods  The neural circuit insult was performed in guinea pigs by rectal transection and subsequent end‐to‐end one layer anastomosis. A 5‐HT₄‐receptor agonist, mosapride citrate (10–100 μmol L⁻¹) (applied for a patent) was applied locally at the anastomotic site.

Key Results  Mosapride promoted the regeneration of the neural circuit in the impaired myenteric plexus and the recovery of the defecation reflex in the distal gut. Furthermore, mosapride generated neurofilament (NF)‐, 5‐HT₄‐receptor‐ and 5‐bromo‐2′‐deoxyuridine (BrdU)‐positive cells and surprisingly formed neural network in the newly formed granulation tissue at the anastomotic site 2 weeks after enteric nerve circuit insult. Possible neural stem cell markers, anti‐distal less homeobox 2 (DLX2)‐ and p75‐positive and NF‐positive cells increased during the same time period. All actions by mosapride were inhibited by the specific 5‐HT₄‐receptor antagonist, GR113808 (10 μmol L⁻¹).

Conclusions & Inferences  These results indicate that activation of enteric neural 5‐HT₄‐receptors promotes reconstruction of an enteric neural circuit leading to the recovery of the defecation reflex in the distal gut, and that this reconstruction involves possibly neural stem cells. These findings indicate that treatment with 5‐HT₄ agonists could be a novel therapy for generating new enteric neurons to rescue aganglionic gut disorders.