Identifying Types of Neurons in the Human Colonic Enteric Nervous System

Distinguishing and characterising the different classes of neurons that make up a neural circuit has been a long-term goal for many neuroscientists. The enteric nervous system is a large but moderately simple part of the nervous system. Enteric neurons in laboratory animals have been extensively characterised morphologically, electrophysiologically, by projections and immunohistochemically. However, studies of human enteric nervous system are less advanced despite the potential availability of tissue from elective surgery (with appropriate ethics permits). Recent studies using single cell sequencing have confirmed and extended the classification of enteric neurons in mice and human, but it is not clear whether an encompassing classification has been achieved. We present preliminary data on a means to distinguish classes of myenteric neurons in specimens of human colon combining immunohistochemical, morphological, projection and size data on single cells. A method to apply multiple layers of antisera to specimens was developed, allowing up to 12 markers to be characterised in individual neurons. Applied to multi-axonal Dogiel type II neurons, this approach demonstrated that they constitute fewer than 5% of myenteric neurons, are nearly all immunoreactive for choline acetyltransferase and tachykinins. Many express the calcium-binding proteins calbindin and calretinin and they are larger than average myenteric cells. This methodology provides a complementary approach to single-cell mRNA profiling to provide a comprehensive account of the types of myenteric neurons in the human colon.