Multidimensional characterization of the physiological and behavioral effects of TCB-2 in mice

Background and PurposeSerotonergic psychedelics affect behavior and physiology, but the relationships among these effects remain poorly understood. In rodents, the head-twitch response is used as a measure of psychedelic-like activity, yet it does not capture changes in physiological state or the performance of learned behaviors. Here, we investigated the acute effects of the 5-HT2A receptor agonist TCB-2 across several behavioral and physiological measures and examined how these effects were modified by pretreatment with the 5-HT2A receptor antagonist volinanserin. Experimental ApproachMice were tested in head-fixed and freely moving conditions. During a learned auditory trace-conditioning task, we measured licking, pupil area, eye position, and blinking. We measured locomotor activity in an open field and quantified head-twitch responses using a DeepLabCut-based method. To examine the contribution of 5-HT2A receptors, mice were pretreated with the 5-HT2A receptor antagonist volinanserin. Key ResultsTCB-2 caused pupil constriction without detectable changes in eye position or blinking when administered alone. TCB-2 also reduced licking at the highest dose, but the cue-locked temporal pattern of licking remained evident. In freely moving mice, TCB-2 reduced locomotor activity and produced a dose-dependent increase in head-twitch responses. Volinanserin partially attenuated TCB-2-induced pupil constriction and reduced head-twitch responses under some conditions, but it did not consistently prevent the other effects of TCB-2. Conclusions and ImplicationsTCB-2 produced distinct effects across physiological and behavioral measures rather than a uniform disruption of behavioral function. Pronounced pupil constriction and head-twitch responses occurred without detectable changes in eye position or blinking, while the temporal organization of conditioned licking was retained despite a reduction in its magnitude. The incomplete and variable effects of volinanserin preclude definitive conclusions about the receptor mechanisms underlying each response. Combining automated head-twitch detection with physiological and task-related measurements provides a broader framework for comparing the pharmacological profiles of serotonergic compounds. What is already knownO_LIClassical psychedelics produce characteristic effects primarily through serotonin 5-HT2A receptor activation. C_LIO_LIHead-twitch responses capture only one dimension of psychedelic-like drug action. C_LI What this study addsO_LITCB-2 reduced locomotion and licking while preserving the cue-locked pattern of conditioned licking. C_LIO_LIPupil constriction occurred without detectable changes in eye position or blinking. C_LI Clinical significanceO_LIMultidimensional phenotyping can distinguish the physiological and behavioral profiles of serotonergic compounds. C_LIO_LIComplementary measures may improve preclinical evaluation of emerging serotonergic therapeutics. C_LI