Evaluation of the total number of myenteric neurons in the developing chicken gut using cuprolinic blue histochemical staining and neurofilament immunocytochemistry

The aim of this study was to find an improved method with which to stain th e entire population of myenteric neurons in the different segments of the d eveloping chicken intestine. Histochemical staining with cuprolinic blue (q uinolinic phthalocyanine) and immunostaining against neurofilament (NF) wer e performed on whole mounts prepared from intestinal segments of embryonic (day 19 of incubation) and hatched (1, 2, 4 and 7 days after hatching) chic kens. Double labelling was performed to evaluate to what extent the two mar kers visualise the same nerve cell population. Cuprolinic blue stained neur onal somata highly selectively, whereas processes and glia cells were poorl y labelled. The cuprolinic blue-positive neurons were uniform in shape. NF immunostaining revealed a morphologically highly variable neuron population . Double labelling with cuprolinic blue and NF resulted in an intensificati on of both stainings, allowing an accurate morphological classification of NF-stained myenteric neurons. Data obtained from the counting of cuprolinic blue-positive neurons were subjected to two-way ANOVA and the Tukey probe. The densities of ganglia and neurons were found,to decrease, and the mean number of neurons per myenteric ganglion to increase, with different dynami cs along the longitudinal axis of the gut during the examined time span. Th e variances in the number of NF-positive neurons were not homogeneous, and the data were therefore not suitable for ANOVA. Accordingly, only semiquant itative conclusions could be drawn.