An accurate method to count human enteric neurons is essential to develop a comprehensive account of the classes of nerve cells responsible for gut function and dysfunction. The majority of cells in the enteric nervous system utilize acetyl choline, or nitric oxide, or a combination of these, as neurotransmitters. Antisera raised against the RNA-binding protein Hu, were used to identify nerve cell bodies in whole mounts of the myenteric plexus of human colon, and then were utilized to analyse cells immunoreactive for combinations of choline acetyltransferase and nitric oxide synthase. Antisera to Hu provided a reliable means to count apparently all enteric nerve cell bodies, revealing 10% more cell bodies than labelling with neuron specific enolase, and no labelling of glial cells as revealed by S100. ChAT+/NOS- neurons accounted for 48% (+/-3%) of myenteric neurons and ChAT-/NOS+ neurons accounted for 43% (+/-2.5%). ChAT+/NOS+ neurons comprised 4% (+/-0.5) of the total number of neurons, and a novel class of small ChAT-/NOS- neurons, making up 5% (+/-0.9%) of all cells, was described for the first time.

There are differences in the structure and function between regions of the colon. In patients with slow transit constipation the activity of all regions is markedly slowed. Counts of colonic neurones in slow transit constipation have been semiquantitative and led to varying results. We have applied new methods of quantification of markers in whole mounts of the colonic myenteric plexus to compare density of innervation between regions and between normal patients and those undergoing resection for severe constipation. Whole mounts of colonic myenteric plexus were made from specimens removed for cancer treatment (controls) and cases of severe constipation. All neurones were labelled by anti-human neuronal protein antibodies. Neurones synthesizing acetyl choline were labelled for choline acetyltransferase (ChAT) and those for nitric oxide by antisera to nitric oxide synthase (NOS). Four populations of neurones were distinguished and quantified according to the two selective markers, ChAT and NOS. In the normal major populations were NOS alone (51% of ascending colon neurones and 44% of descending colon neurones) and ChAT alone (41% ascending colon, 48% descending colon). Nitric oxide synthase/ChAT and NOS-/ChAT-comprised only small populations. In all regions in severe constipation, the percentage of NOS-only colonic myenteric neurones was raised (54% ascending colon, 49% descending colon) and ChAT only was reduced (36% ascending colon, 42% descending colon). The other populations were not changed. Accurate quantification of neuronal populations in whole mounts of human colon reveals inter-regional differences in innervation and marked changes in innervation in cases of very severe constipation.

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