Hierarchical and Spatial Mapping of Whole-Brain c-Fos Activity Reveals Distinct Opioid and Withdrawal Neuronal Ensembles

How opioid exposure and withdrawal states shape brain activity at the systems and circuit level remains poorly understood. Here, we use whole-brain, cellular-resolution c-Fos mapping to define brain-wide activity patterns and neuronal ensembles associated with morphine administration and withdrawal. To account for the brain’s anatomically nested structure, we developed and applied a hierarchical statistical framework that detects region-specific changes in activity and outperforms conventional methods that treat brain regions as individual, unrelated units. These distributed signals formed ensembles with consistent and anatomically structured patterns of activity, both within subregions and across multiple connected brain areas. By combining TRAP2-based activity tagging with acute whole-brain c-Fos staining, we identified morphine- and withdrawal-activated ensembles and found that they are largely non-overlapping at the single-cell level, even within the same brain region. Integration with existing spatial transcriptomics datasets identified molecular markers for these state-specific ensembles in key brain areas such as the nucleus of accumbens, amygdala and ventral tegmental areas. Lastly, by integrating Allen mouse whole-brain transcriptional datasets, we identified the molecular identity of the morphine- administration and withdrawal ensembles. These findings define dissociable neuronal ensembles that encode opposing drug states and introduce a scalable framework for linking whole-brain activity to molecular and circuit-level mechanisms.