Full thickness three-dimensional mapping of neuronal populations within the avian enteric nervous system

The enteric nervous system (ENS) serves as the principal regulator of intestinal muscular activity in the poultry gastrointestinal tract, modulating secretory processes and digestive functions, while also contributing to immune protection at the muscularis level against pathogen infections. While ENS structure and function are well studied in mammals, knowledge of the poultry ENS remains limited. In this study, we provide the first comprehensive characterization of neuronal populations and structural organization within the three‑dimensional environment of the chicken ENS using tissue clearing and 3D imaging without sectioning. Full-thickness preparations of ileum, cecum, and colon from 29‑day‑old chickens were fixed in 4% paraformaldehyde and stained for HuC/D, a pan‑neuronal cell body marker, and neuronal nitric oxide synthase (nNOS), a marker for nitrergic inhibitory neurons and connecting fibers. Tissue clearing enabled optimal visualization of enteric neuronal cell bodies and fibers in both the myenteric and submucosal plexuses of each intestinal region using confocal microscopy. The chicken ENS exhibits clear regional specialization, with higher ganglion density in the ileum and colon than in the cecum (p < 0.05). The submucosal plexus displays higher neuronal density and contains more ganglia than the myenteric plexus (p < 0.05), underscoring its central role in regulating the intense digestive activity driven by continuous feeding. Notably, the cecum shows a higher proportion of nitrergic neurons (p < 0.05), suggesting enhanced nitrergic-mediated inhibitory control of slow segmental motility that supports microbially mediated fermentation and fluid retention. Our findings establish a novel staining and clearing approach that preserves the spatial organization ENS units across the chicken intestinal wall. This methodology provides a valuable platform for poultry researchers to investigate the functional contribution of the ENS in poultry gut health.