An Anterior Cingulate Cortex Neuronal Ensemble Controls Contextual Opioid Analgesic Tolerance

Abstract Opioids are first-line treatments for pain; however, tolerance leads to decreased efficacy and escalated dosing, increasing the risk of opioid use disorder and overdose. Despite well-established cellular and molecular adaptations, opioid analgesic tolerance can be rapidly reversed in settings where these drugs are not expected. The specific neuronal populations that orchestrate this expectation-based tolerance are not known. In this study, we used a contextual tolerance training method, whole-brain clearing, and immunostaining to identify brain regions involved in contextual tolerance and to pinpoint a neuronal ensemble in the anterior cingulate cortex (ACC) that is activated by contextual analgesic tolerance. We observed that calcium activity in principal neurons of the ACC is suppressed by fentanyl in opioid-naïve mice and during contextual reversal, but not during contextual tolerance. Chemogenetic silencing of the ACC induced analgesic tolerance reversal in the opioid-associated context without affecting thermal nociception in opioid-free mice. Conversely, chemogenetic activation of the ACC contextual tolerance-active neuronal ensemble triggered analgesic tolerance in an unassociated context. This research highlights the role of ACC neuronal ensembles in mediating expectation-driven, contextual opioid analgesic tolerance without affecting basal nociception. Modulating ACC activity could provide a promising strategy to improve pain relief while maintaining the essential ability to detect harmful stimuli. Competing Interest Statement The authors have declared no competing interest. Footnotes The authors have declared that no conflict of interest exists. Add a new figure showing anterior cingulate cortex activity during hot plate testing Add more replicate to existing figure 5 - chemogenetic experiments Add control experiments in supplemental figures 1 and 2 Data Availability The full dataset from this study is detailed in the main paper or the supplemental material, with raw data accessible in the Supporting Data Values file.