Cortico-subcortical dysconnectivity following opioid administration correlates with analgesia in the awake mouse brain

The µ-opioid receptor (MOP) plays a pivotal role in mediating both the therapeutic effects and abuse potential of opioid drugs. However, the comprehensive understanding of the large-scale brain effects resulting from MOP activation remains elusive. In this study, we introduce a multimodal experimental pipeline combining awake functional ultrasound (fUS) imaging through the intact skull with behavioural and molecular measurements to assess the dynamics and physiological relevance of opioid-induced changes in brain activation and functional connectivity (FC) patterns. Application of the major opioid drugs morphine, fentanyl, methadone, and buprenorphine induces a robust, dose- and time-dependent reorganization of brain activation and FC patterns in awake and behaving mice. In addition to a rapid and transitory region-specific opioid-induced hyperperfusion, a slower and highly consistent MOP-specific dysconnectivity fingerprint emerges, which is sensitive to induced tolerance or to pharmacological and genetic MOP inactivation, characterized by decreased FC of the somatosensory cortex to hippocampal and thalamic regions. Simultaneously, subcortical FC and bilateral FC of the somatosensory cortex are increased. Analysis of underlying dynamics reveal significant changes in intra- and inter-regional oscillation synchronicity, leading to opioid-induced loss of critical-state dynamics. Notably, rapid local perfusion changes are correlated with hypermobility and respiratory depression, while slower FC changes, displaying spatio-temporal profiles specific to agonists, correlate with generalized brain MOP activation and the development of analgesia. Our results highlight the reorganization of inter-regional FC as a prominent brain effect of opioids and emphasize the potential of our approach in identifying novel neuropsychiatric drugs with improved pharmacological profiles.