Central amygdalar PKCδ neurons mediate fentanyl withdrawal

Aversion to opioid withdrawal is a significant barrier to achieving lasting opioid abstinence. The central amygdala (CeA), a key brain region for pain, threat-detection, autonomic engagement, and valence assignment, is active during opioid withdrawal. However, the role of molecularly distinct CeA neural populations in withdrawal remains underexplored. Here, we investigated the activity dynamics, brain-wide connectivity, and functional contribution of Protein Kinase C-delta (PKCδ)-expressing neurons in the CeA lateral capsule (CeLC ^PKCδ ) during fentanyl withdrawal in mice. Mapping activity-dependent gene expression in CeLC ^PKCδ neurons revealed a highly withdrawal-active subregion in the anterior half of the CeA. Fiber photometry calcium imaging showed that opioid-naïve CeLC ^PKCδ neurons respond to salient noxious and startling stimuli. In fentanyl-dependent mice, naloxone-precipitated withdrawal increased spontaneous neural activity and enhanced responses to noxious stimuli. Chronic inhibition of CeLC ^PKCδ neurons throughout fentanyl exposure, via viral overexpression of the potassium channel Kir2.1, attenuated withdrawal signs in fentanyl-dependent mice. Lastly, we identified putative opioid-sensitive inputs to CeLC ^PKCδ neurons using rabies-mediated monosynaptic circuit tracing and color-switching tracers to map mu-opioid receptor-expressing inputs to the CeLC. Collectively, these findings suggest that the hyperactivity of CeLC ^PKCδ neurons underlies the somatic signs of fentanyl withdrawal, offering new insights into the amygdala cell-types and circuits involved in opioid dependence.