Abstract
<title>Abstract</title> <p> <italic>Rationale.</italic> Fentanyl (FEN) is a highly potent opioid and major contributor to opioid-related deaths. Sex differences in responses to psychoactive drugs, including opioids, are well-documented and influenced by circulating hormones. Pain elicits sensory and affective responses measurable through ultrasonic vocalizations (USVs) in rats. Although this affective aspect contributes to pain distress, few studies examine whether sex or hormonal state modulates it. <italic>Objectives</italic> . We examined whether sex, estrous stage (proestrus vs. non-proestrus), or FEN differentially alter affective and sensory responses to a pain stimulus and neural activation in pain-associated regions including the periaqueductal gray (lPAG), dorsal raphe nuclei (DRN), and amygdala. <italic>Methods.</italic> Sprague-Dawley male and female (proestrus or non-proestrus) rats were tested for: (1) FEN-induced locomotor activity and USV responses; (2) FEN-induced sensory and affective analgesic responses to tail flick; and (3) neural activation to tail flick through cFos expression. <italic>Results.</italic> Females showed greater and more prolonged activity levels and emitted more USVs at the lower FEN dose (0.05 mg/kg) with no estrous stage effect. Yet, males showed stronger sensory analgesic responses to FEN (0.05 mg/kg) but emitted fewer USVs. Tail-flick induced cFos expression but did not differ by sex or estrous stage. Tail flick latency positively predicted IPAG and DRN cFos expression and 55 kHz USVs negatively predicted IPAG activation. <italic>Conclusions.</italic> These findings suggest that sex differences in opioid responses are dissociable across sensory and affective domains, with more stable sex differences in sensory processing and more variable sex differences in affective responding that are minimally influenced by estrous phase. </p>