Myenteric neuron numbers are maintained in aging mouse distal colon

Background Age‐associated myenteric neuronal loss has been described in several species. In some studies, cholinergic neurons have been reported to be selectively vulnerable, whereas nitrergic neurons are spared. Aging of the mouse enteric nervous system (ENS) and the subtypes of mouse myenteric neurons that may be lost have been little studied. We therefore investigated changes in the numbers of total neurons and two neuronal subpopulations in the mouse distal colon during aging. Methods Wholemount preparations from 3–4‐, 12–13‐, 18–19‐, and 24–25‐month‐old C57BL/6 mice were double immunolabeled with HuC/D antibody to identify the total neuronal population and antisera to either calbindin or neuronal nitric oxide synthase (nNOS) to identify myenteric neuronal subpopulations. Samples were analyzed by confocal microscopy. New procedures were employed to ensure unbiased counting and to correct for changes in gut dimensions with age and stretch during sample preparation. The density of nerve fibers in the tertiary plexus was also studied. Key Results No significant change in numbers of total neurons or of either subpopulation with age was measured, but because of gut growth, the density of myenteric neurons decreased between 3–4 and 12–13 months. The density of nNOS‐immunoreactive nerve fibers in the tertiary plexus increased significantly with age, up to 18–19 months. Numerous swollen processes of CB and nNOS‐immunoreactive neurons were observed in 18–19‐ and 24–25‐month‐old animals. Conclusions & Inferences These results indicate that aging does not result in a loss of myenteric neurons in mouse distal colon at the ages studied, although neurodegenerative changes, which may impact on neuronal function, do occur.