5‐Hydroxytryptamine (5‐HT, serotonin) is an important regulator of colonic motility and secretion; yet the role of serotonergic neurons in the colon is controversial.

We used immunohistochemical techniques to examine their projections throughout the enteric nervous system and interstitial cells of Cajal (ICC) networks in the murine proximal to mid colon.

Serotonergic neurons, which were mainly calbindin positive, occurred only in myenteric ganglia (1 per 3 ganglia). They were larger than nNOS neurons but similar in size to Dogiel Type II (AH) neurons. 5‐HT neurons, appeared to make numerous varicose contacts with each other, most nNOS neurons, Dogiel Type II/AH neurons and glial cells. 5‐HT, calbindin and nNOS nerve fibers also formed a thin perimuscular nerve plexus that was associated with ganglia, which contained both nNOS positive and negative neurons, which lay directly upon the submucosal pacemaker ICC network. Neurons in perimuscular ganglia were surrounded by 5‐HT varicosities. Submucous ganglia contained nNOS positive and negative neurons, and calbindin positive neurons, which also appeared richly supplied by serotonergic nerve varicosities. Serotonergic nerve fibers ran along submucosal arterioles, but not veins. Varicosities of serotonergic nerve fibers were closely associated with pacemaker ICC networks and with intramuscular ICC (ICC‐IM). 5‐HT_2B receptors were found on a subpopulation of non‐5‐HT containing myenteric neurons and their varicosities, pacemaker ICC‐MY and ICC‐IM.

Myenteric serotonergic neurons, whose axons exhibit considerable divergence, regulate the entire enteric nervous system and are important in coordinating motility with secretion. They are not just interneurons, as regularly assumed, but possibly also motor neurons to ICC and blood vessels, and some may even be sensory neurons.