Abstract: Objective To establish a mouse model with specific knockout of dopamine D2 receptor (D₂R) in the enteric nervous system (ENS) and elucidate its mechanism in promoting intestinal motility. Methods The Cre-LoxP system was employed to generate ENS-specific D₂R knockout mice (D₂R^Nmu-CKO) and control mice (D₂R^flox/flox). Expression levels of D₂R and choline acetyltransferase (ChAT) were detected using immunofluorescence staining, DAB staining, RT-qPCR, and Western blot. Acetylcholine (ACh) content was measured by ELISA. Intestinal transit was assessed in vivo using X-ray imaging in live small animals. Results Immunofluo- rescence staining confirmed the co-localization of D₂R with the neuronal marker PGP 9.5 as well as the cholinergic neuronal marker ChAT in the duodenal myenteric plexus (MP) of wild-type mice, indicating that D₂R is expressed in cholinergic neurons of the duodenal ENS. Genotyping confirmed successful establishment of D₂R^Nmu-CKO mice (genotypes: Nmu-Cre^+/+D₂R-flox^+/+ or Nmu-Cre^+/-D₂R-flox^+/+) and control D₂R^flox/flox mice (genotype: Nmu-Cre^-/- D₂R-flox^+/+). Compared with control mice, D₂R^Nmu-CKO mice exhibited significantly reduced mRNA(n=6, P＜0.001) and protein (n=8, P＜0.01) expression of D₂R in the duodenal muscle layer, while mRNA(n=6, P＜0.001) and protein (n=6, P＜0.05) expression of ChAT were markedly increased. The content of ACh was also significantly elevated (n=8, P＜0.01). DAB staining further revealed a significant increase in the number of ChAT-positive neurons in the duodenal MP of D₂R^Nmu-CKO mice (n=6, P＜0.01). Intestinal transit assays demonstrated accelerated intestinal transit and significantly shortened whole-gut transit time in D₂R^Nmu-CKO mice (n=5, P＜0.05). Conclusions This study successfully established an ENS-specific D₂R knockout mouse model and revealed that loss of ENS D₂R promotes intestinal transit by enhancing ACh release. These findings provide a theoretical basis for novel strategies targeting ENS D₂R to treat gastrointestinal motility disorders such as functional dyspepsia and slow-transit constipation.

Key words: enteric nervous system, dopamine D2 receptor, acetylcholine, intestinal transit