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.

UCHL1 (ubiquitin carboxyterminal hydrolase 1) is a deubiquitinating enzyme that is particularly abundant in neurons. From studies of a spontaneous mutation arising in a mouse line it is clear that loss of function of UCHL1 generates profound degenerative changes in the central nervous system, and it is likely that a proteolytic deficit contributes to the pathology. Here these effects were found to be recapitulated in mice in which the Uchl1 gene had been inactivated by homologous recombination. In addition to the previously documented neuropathology associated with loss of UCHL1 function, axonal swellings were detected in the striatum. In agreement with previously reported findings the loss of UCHL1 function was accompanied by perturbations in ubiquitin pools, but glutathione levels were also significantly depleted in the brains of the knockout mice, suggesting that oxidative defense mechanisms may be doubly compromised. To determine if, in addition to its role in the central nervous system, UCHL1 function is also required for homeostasis of the enteric nervous system the gastrointestinal tract was analyzed in UCHL1 knockout mice. The mice displayed functional changes and morphological changes in gut neurons that preceded degenerative changes in the brain. The changes were qualitatively and quantitatively similar to those observed in wild type mice of much greater age, and strongly resemble changes reported for elderly humans. UCHL1 knockout mice should therefore serve as a useful model of gut aging.

The density of immunoreactivity of markers for some types of IAS nerve fibers decreases during aging, which may contribute to age-related ano-rectal dysfunction.

Ageing is associated with impaired neuromuscular function of the terminal gastrointestinal (GI) tract, which can result in chronic constipation, faecal impaction and incontinence. Interstitial cells of cajal (ICC) play an important role in regulation of intestinal smooth muscle contraction. However, changes in ICC volume with age in the terminal GI tract (the anal canal including the anal sphincter region and rectum) have not been studied. Here, the distribution, morphology and network volume of ICC in the terminal GI tract of 3‐ to 4‐month‐old and 26‐ to 28‐month‐old C57BL/6 mice were investigated. ICC were identified by immunofluorescence labelling of wholemount preparations with an antibody against c‐Kit. ICC network volume was measured by software‐based 3D volume rendering of confocal Z stacks. A significant reduction in ICC network volume per unit volume of muscle was measured in aged animals. No age‐associated change in ICC morphology was detected. The thickness of the circular muscle layer of the anal sphincter region and rectum increased with age, while that in the distal colon decreased. These results suggest that ageing is associated with a reduction in the network volume of ICC in the terminal GI tract, which may influence the normal function of these regions.