Psychedelics and psychostimulants produce subjective and often positive affective effects in humans. In rats, positive affective states are often associated with frequency-modulated ultrasonic vocalizations (USVs). We examined whether the hallucinogenic 2,5-dimethoxy-4-iodoamphetamine (DOI; 1.25-2.5 mg/kg) and LSD (0.04-0.08 mg/kg), the non-hallucinogenic lisuride (0.03 or 0.3 mg/kg) and the psychostimulant amphetamine (1 mg/kg) modulate USV emission, activity, and head-twitch response (HTR) in male Wistar rats. USVs were detected using DeepSqueak and clustered in a custom, unsupervised manner using variational autoencoder-based, and frequency-based embeddings. Behavior was analyzed using DeepLabCut and SimBA AI/machine learning tools. DOI (2.5 mg/kg) and LSD (0.08 mg/kg), but not lisuride or amphetamine induced HTR, whereas only amphetamine (1 mg/kg) increased activity. DeepSqueak detected nearly 48,000 calls, and UMAP-based k-means clustering identified four broad call categories: 22-kHz alarm and 50-kHz flat, step-up/trill, and upward-ramp-like calls. Total call emission increased after amphetamine and both DOI doses. However, cluster-based analyses indicated that the compounds did not merely increase overall calling but altered the vocal repertoire. DOI and LSD (0.04 mg/kg) reduced occupancy of the flat-call cluster, a call type typically unrelated to reward or appetitive behavior. In contrast, LSD (0.08 mg/kg), lisuride (0.03 mg/kg), and amphetamine increased occupancy of frequency-modulated clusters, call categories often associated with positive affect. Serotonergic psychedelics, lisuride, and amphetamine alter rat ultrasonic vocalizations and may shift vocal repertoires toward patterns associated with positively valenced states. USV profiles did not parallel HTR and should be viewed as a complementary affective readout rather than a substitute marker of psychedelic-like activity.
