5-HT ₄ receptor ligand RS67333 modulates striatal acetylcholine and dopamine release via inhibition of acetylcholinesterase

Serotonin 5-HT4 receptors (5-HT ₄ Rs) have emerged as potential therapeutic targets in neuropsychiatric and neurodegenerative disorders by modulating circuits that shape mood, cognition, and motor function. Ligands for 5-HT ₄ Rs can modify dopamine (DA) and acetylcholine (ACh) transmission but mechanisms and circuits have not been fully resolved. Some 5-HT ₄ R agonists have been suggested to have effects that include inhibition of acetylcholinesterase (AChE), raising speculation that 5-HT ₄ R ligands might modulate ACh and/or DA through this action. Here, we investigated the impact of RS67333, a partial 5-HT ₄ R agonist, on DA and ACh release dynamics in the striatum detected ex vivo in mouse brain slices using fast-scan cyclic voltammetry and genetically encoded ACh sensor GRAB _ACh3.0 respectively. We found that RS67333 significantly modulated electrically evoked DA release in dorsolateral striatum and nucleus accumbens core, effects that were abolished by a nicotinic receptor (nAChR) antagonist. In parallel, RS67333 altered evoked ACh signals by extending extracellular ACh lifetime, and correspondingly, RS67333 was found to inhibit striatal AChE enzymatic activity. By contrast, BIMU8, an alternative 5-HT ₄ R ligand that did not inhibit striatal AChE, had no effect on evoked striatal ACh or DA release. These findings indicate that RS67333 modulates striatal ACh transmission, which shapes downstream regulation of DA release by nAChRs, not through 5-HT ₄ Rs but through AChE inhibition. These findings emphasize the caution due in attributing functions to 5-HT ₄ Rs, but also highlight an alternative pharmacological profile of some purported 5-HT ₄ R ligands as AChE inhibitors of potential utility for treating ACh/DA disorders.