Evaluation of the activity of chemically identified enteric neurons through the histochemical demonstration of cytochrome oxidase

The measurement of the density of the reaction product produced by the histochemical demonstration of cytochrome oxidase activity provides a method for the visual identification of physiologically active enteric neurons. The current study utilized the cytochrome oxidase technique in order to evaluate the metabolic history of neurons in different regions of the bowel and in chemically identified types of neuron. In addition, the effect of drugs or neurotoxins commonly used in the immunocytochemical identification of enteric neuronal phenotypes was also analyzed. Cytochrome oxidase activity was visualized with a blue‐black reaction product resulting from the cobalt‐intensified oxidation of 3,3′‐diaminobenzidine. Peptides or 5—hydroxytryptamine (5‐HT) were localized with biotinylated secondary antibodies and alkaline phosphatase‐labeled avidin. Bound avidin or endogenous alkaline phosphatase was visualized with a red reaction product in the presence or absence, respectively, of levamisole. Use of monochromatic red light permitted the cytochrome oxidase reaction product density to be measured without interference from a simultaneously demonstrated histo‐or immunochemical marker. A multi‐peptidergic class of cholinergic submucosal secretomotor neuron containing neuropeptide Y (NPY) and calcitonin gene related peptide (CGRP)immunoreactivities was found to be less metabolically active than the average of all submucosal neurons. In contrast, a non‐cholinergic submucosal secretomotor neuron containing dynorphin (which is also known to contain vasoactive intestinal peptide) immunoreactivity was more metabolically active than submucosal neurons that do not contain this peptide. On average, submucosal neurons were more metabolically active than those of the myenteric plexus, and levels of metabolic activity in the myenteric plexus were found to be higher in the duodenum and the cecum than in the jejunum‐ileum or colon. Myenteric neurons characterized by CGRP or NPY immunoreactivities or by endogenous alkaline phosphatase activity, were all less metabolically active than the average of all neurons in myenteric ganglia. Colchicine, which stimulates intestinal motility, was observed to increase cytochrome oxidase activity in enteric neurons, suggesting that an effect on the enteric nervous system contributes to its action on the bowel. The neurotoxins, 6‐hydroxydopamine and 5,7‐dihydroxytryptamine (5,7‐DHT) were each found to stimulate neuronal metabolic activity. 5,7‐DHT appeared to activate excitatory subtypes of 5‐HT receptor since its effects were blocked or mimicked by compounds that act as antagonists or agonists, respectively, at these receptors. The cytochrome oxidase technique is thus useful for studying the metabolic activity of chemically identified types of enteric neuron as well as for evaluating the action of experimental procedures on these cells.