Cholinergic innervation in the developing chick cloaca and colorectum.

PURPOSE: Acetylcholine is the major excitatory neurotransmitter involved in intestinal contraction and therefore plays a pivotal role in normal gastrointestinal motility. Acetylcholinesterase (AchE) is an enzyme involved in hydrolysing acetylcholine during its metabolism. The AchE histochemistry can therefore be used to label acetylcholine-positive nerve cells and fibres, giving an overview of cholinergic innervation in the gut. Persistent cloaca is the most common anorectal malformation seen in female infants. The normal avian embryo contains a cloaca, reminiscent to the human malformation, which acts as a collecting chamber into which the digestive, urinary, and reproductive tracts merge. The aim of our study was to investigate cholinergic innervation in the chick embryo cloaca and colorectum at various stages of development.

METHODS

Chick embryos were harvested at embryonic days 12 (E12), E14, E16, and E18. The colorectum and cloaca were removed from each embryo and frozen. Frozen transverse sections were then cut and AchE histochemistry was performed. The AchE staining was evaluated and graded using light microscopy.

RESULTS

Results of our study show that acetylcholine is increasingly expressed in the chick embryo cloaca and colorectum from E12 to E18, with a stronger expression in the colorectum at each stage. The AchE-positive ganglia and fibres were evident in the submucosal and myenteric plexuses at all stages in both the cloaca and the colorectum. Ganglia size was seen to increase with age, and there was a notable increase in the amount of AchE-positive nerve fibres.

CONCLUSION

Results show an increase in cholinergic innervation in both the cloaca and colorectum during development. As acetylcholine is the major excitatory neurotransmitter in the enteric nervous system, this study provides us with valuable information regarding the regional differences in the development of cholinergic innervation of the embryonic hindgut.