5-HT4 receptor ligand RS67333 modulates striatal acetylcholine and dopamine release via inhibition of acetylcholinesterase

ABSTRACT Serotonin 5-HT4 receptors (5-HT4Rs) have emerged as potential therapeutic targets in neuropsychiatric and neurodegenerative disorders by modulating circuits that shape mood, cognition, and motor function. Ligands for 5-HT4Rs can modify dopamine (DA) and acetylcholine (ACh) transmission but mechanisms and circuits have not been fully resolved. Some 5-HT4R agonists have been suggested to have off-target, pleiotropic effects that include inhibition of acetylcholinesterase (AChE), raising speculation that 5-HT4R ligands might modulate ACh and/or DA through this action. Here, we investigated the impact in the striatum of RS67333, a partial 5-HT4R agonist, on DA and ACh release dynamics detected ex vivo in mouse brain slices using fast-scan cyclic voltammetry and genetically encoded ACh sensor GRABACh3.0 respectively. We found that RS67333 significantly modulated electrically evoked DA release in dorsolateral striatum (DLS) and nucleus accumbens core (NAcC), effects that were abolished by a nicotinic receptor (nAChR) antagonist. In parallel, RS67333 altered evoked ACh signals in DLS and NAcC by extending extracellular ACh lifetime, and correspondingly, RS67333 inhibited striatal AChE enzymatic activity detected in a chemiluminescent assay. By contrast, BIMU8, a 5-HT4R ligand that lacked AChE inhibitory properties, had no effect on evoked striatal ACh or DA release. These findings reveal that RS67333 acts through AChE inhibition, and not 5-HT4Rs, to modulate striatal ACh transmission, which shapes downstream regulation of DA release by nAChRs. These findings emphasize the caution that should be taken in identifying 5-HT4R function using ligands alone, but also highlight a pharmacological profile of RS67333 of potential benefit to DA/ACh disorders. FILE DESCRIPTIONS This repository contains the following files: Key Resources Table (.xlsx) - Table containing details on key resources (mouse lines, virus strains, and software), and the persistent identifiers for protocols and code used and generated in this study. Source Data Folder (.zip): _README_Source_Data (.txt) with detailed information about each dataset. Individual tabular datasets corresponding to panels shown in the Main Figures 1 to 4 (.csv). Excel spreadsheet containing all tabular datasets plotted in Main Figures 1 to 4 (.xlsx)