Following decades of prohibition, psychedelic drugs have reemerged as promising therapeutics for stress-related conditions, including depression and post-traumatic stress disorder. Still, their impact on stress-related brain regions and the hypothalamic-pituitary-adrenal (HPA) axis remains unclear. This work explores the acute effects of psilocybin on the primary regulators of the HPA axis: corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus (CRH). Here, using blood plasma measurements and in vivo single-fiber photometry, we demonstrate that psilocybin induces robust activation of the HPA axis via CRHneurons, with more pronounced responses observed in female mice and a reliance on serotonergic 5-HT2A and 5-HT2C receptors. Ex vivo electrophysiology indicates that the 5-HT2A-receptor-mediated effects involve dual mechanisms: direct post-synaptic depolarization of CRHneurons and increased presynaptic glutamate release. Our findings also reveal that psilocybin alters how CRHneurons react to environmental changes, resulting in a surprising decrease in activity that contrasts with typical elevated stress responses. This context-specific modulation may be a key mechanism underlying the therapeutic potential of psychedelics to recalibrate maladaptive stress reactivity. Our findings emphasize the interplay between the serotonergic and stress systems and support the considerable influence of contextual factors, i.e., "setting," on the psychedelic experience. This study provides the first real-time in vivo evidence of neuronal activation of the stress system following psilocybin administration and has significant implications for optimizing the therapeutic efficacy of psychedelic-assisted therapy. PVN PVN PVN PVN
