Varenicline as a DREADD Actuator Elicits Off-Target Effects on Somatosensation, Locomotion, and Anxiety-Related...

As a new-generation chemogenetic actuator in designed receptors exclusively activated by designer drugs (DREADD) systems, varenicline (VAR) is gaining popularity in studies involving both nonhuman primates and rodents. However, its acute off-target effects on somatosensation and anxiety-related behaviors in rodents remain largely uncharacterized. Using classical behavioral paradigms─including evoked itch-scratching, noxious thermal pain testing, open field, and elevated plus maze assays─we demonstrate that intraperitoneal administration of standard VAR doses modulates itch/pain sensation, basal locomotor activity, and anxiety-related emotional behaviors in Sprague-Dawley (SD) rats. Key findings include the following: high-dose VAR (0.2 mg/kg) significantly increased 5-HT-induced itch-scratching behavior; both low- (0.1 mg/kg) and high-dose VAR elevated sensitivity to noxious thermal stimuli; high-dose VAR enhanced basal locomotor activity in OFT, while low-dose VAR induced anxiety-related phenotypes in EPM. Fiber photometry further revealed that both doses of VAR enhanced neuronal activity in the prelimbic cortex (PrL) and central amygdala (CeA). These results indicate that VAR, at doses commonly employed for DREADD activation, elicits off-target effects on somatosensation, spontaneous locomotion, and anxiety-related emotional behaviors. The underlying mechanisms may involve VAR-induced neuronal hyperactivation in the PrL and CeA, potentially mediated by ligand/metabolite interactions with endogenous brain receptors. By identifying dose-dependent off-target actions of VAR, a next-generation chemogenetic ligand, this study underscores the necessity of including ligand-only controls in DREADD experiments and urges cautious interpretation of behavioral outcomes attributed to VAR-mediated chemogenetic manipulations.