Quantification of neurons in the myenteric plexus: an evaluation of putative pan-neuronal markers

Accurate estimates of the total number of neurons located in the wall of the gut are essential for studies of the enteric nervous system (ENS). Though several stains and antibodies are used routinely as pan-neuronal markers, controversies of relative sensitivity and completeness have been difficult to resolve, at least in part because comparisons often must be made across experiments and laboratories. Therefore, we evaluated the efficacy of four putative pan-neuronal markers for the ENS, under comparable conditions. Neurons in the myenteric plexus of wholemounts taken from the small intestines of Fischer 344 rats were stained using Cuprolinic Blue, anti-HuC/D, anti-protein gene product 9.5, or FluoroGold injections followed by permanent labeling with an antibody to the FluoroGold molecule. All four markers had useful features, but both protein gene product 9.5 and FluoroGold were found to be problematic for obtaining reliable counts. As a result, only neurons labeled with either Cuprolinic Blue or anti-HuC/D were compared quantitatively. Based on counts from permanently labeled tissue, Cuprolinic Blue and HuC/D were similarly effective in labeling all neurons. Because the two protocols have different strengths and weaknesses, Cuprolinic Blue and HuC/D provide a complementary set of labels to study the total neuronal population of the ENS.