DREADDs-Based Chemogenetics Induced Slow Transit Constipation via Inhibition of Enteric Neurons

Designer receptors exclusively activated by designer drugs (DREADDs)-based chemogenetic tools are commonly used to activate or silence targeted neurons by the agonistic ligand deschloroclozapine (DCZ). This study aimed to establish a Gi-DREADD-based murine model of slow transit constipation (STC) and elucidate its pathophysiological mechanisms.

Adeno-associated virus (AAV) 9-hM4Di was injected into the intestinal wall of mice, and colonic motility was evaluated. The efficiency and immunogenicity of AAV9-hM4Di transduction in the enteric nervous system (ENS) were evaluated. Nitric oxide (NO), acetylcholine (ACh), and substance P (SP) in the colonic tissues and serum samples were analyzed. Calcium (Ca²⁺) imaging was performed to evaluate the responses of AAV9-hM4Di on enteric nerves.

AAV9-hM4Di-treated mice showed gastrointestinal motility dysfunction, including reduced fecal pellets and decreased fecal mass and water content. Electrophysiological recording of muscle contraction in the isolated colonic tissues from the chemogenetic mice showed decreased frequency and amplitude after DCZ treatment. The mice treated with AAV9-hM4Di showed the highest levels of transduction in the myenteric plexuses of the ENS. There were no differences in transduction in neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT) neurons. Gi-DREADDs significantly downregulated ACh but not NO or SP expression in the distal colon in the chemogenetic mice. Ca²⁺ transient in neurons of ENS in chemogenetic mice was strongly inhibited by DCZ.

It is feasible to apply the DREADDs-based chemogenetic tools to the ENS. Gi-DREADDs can selectively modulate the ENS, inducing STC without excitatory-neural bias, offering targeted neuromodulation for gastrointestinal motility disorders.