Functional and Structural Investigation of Myenteric Neurons in the Human Colon

Abstract

Background and aims: The enteric nervous system independently controls gastrointestinal function including motility, which is primarily mediated by the myenteric plexus, therefore also playing a crucial role in functional intestinal disorders. Live recordings from human myenteric neurons proved to be challenging due to technical difficulties. Using the neuroimaging technique, we are able to record human colonic myenteric neuronal activity and investigate their functional properties in a large cohort of patients.

Methods: Activity from myenteric neurons in wholemount preparations of different sampling sites of fresh, human colonic tissue was recorded using neuroimaging with the voltage sensitive dye 1-(3-sulfanatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine. Neuronal responses were analyzed following stimulation with nicotine and serotonin (5-HT) for differences based on the donor's age, the disorder indicative for surgery and the colonic region. Immunohistochemistry was performed to calculate the total neuronal numbers.

Results: Stimulation with nicotine and 5-HT elicited reproducible action potential discharge in a proportion of human myenteric neurons. The responses to 5-HT were significantly greater in tissues from older patients and from those with inflammatory disorders, while neuronal activity to nicotinergic stimulation was comparable in all patients. Neuronal numbers declined with rising patient's age and was highest in the sigmoid colon.

Conclusion: Neuroimaging with 1-(3-sulfanatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine was successfully adapted to record reproducible responses from human colonic myenteric neurons upon pharmacological stimulation. Evidence exists for an impact of age and inflammation on the serotonergic neuronal signaling and for differences in neuronal numbers in the distinct colonic regions as well as a neuronal decrease with age.

Keywords: Age; Enteric neurons; Large intestine; Neuroimaging; Serotonin.

Graphical abstract

Figure 1

The image shows one neuron (encircled in blue) in the sigmoid colon responding to the spritz application of serotonin (5-HT). The upper trace corresponds to the marked neuron during the blank recording, the lower trace following the 5-HT application. An exemplary action potential is indicated by the arrow; the grey pipette shows the direction of the 5-HT application. Scale bar = 100 μm.

Figure 2

Human colonic myenteric neuronal activity parameters in response to serotonin (5-HT, 1 mM) application. (A) The number of action potentials (APs) fired in response to 5-HT application significantly increased with rising age of the tissue donor (Spearman correlation, r = 0.4, P = .007). (B) The burst frequencies were significantly higher in tissues from patients with inflammatory than in tissues from patients with cancerous disorders, respectively (unpaired t-test, P = .0029).

Figure 3

Neuronal numbers in the myenteric plexus of the human colon. A: The number of neurons/mm² tissue was significantly higher in the c.s./r. than in the c.a. (Kruskal-Wallis test, P = .002, with Dunn’s multiple comparisons test). B: The number of neurons/mm² ganglionic surface area was significantly higher in the c.t. and c.s./r than in the c.a. (Kruskal-Wallis test, P < .0001, with Dunn’s multiple comparisons test) and declined significantly from the young adults to the adults and to the elderly (Kruskal-Wallis test, P = .006, with Dunn‘s multiple comparisons test). c.a., ascending colon; c.t., transversum colon; c.d., colon descendens; c.s./r, sigmoid colon/rectum.