Anterior cingulate cortex is necessary for spontaneous opioid withdrawal and withdrawal-induced hyperalgesia in male mice

The anterior cingulate cortex (ACC) is implicated in many pathologies, including depression, anxiety, substance-use disorders, and pain. There is also evidence from brain imaging that the ACC is hyperactive during periods of opioid withdrawal. However, there are limited data contributing to our understanding of ACC function at the cellular level during opioid withdrawal. Here, we address this issue by performing ex vivo electrophysiological analysis of thick-tufted, putative dopamine D2 receptor expressing, layer V pyramidal neurons in the ACC (ACC L5 PyNs) in a mouse model of spontaneous opioid withdrawal. We found that escalating doses of morphine (20, 40, 60, 80, and 100 mg/kg, i.p. on days 1-5, respectively) injected twice daily into male C57BL/6 mice evoked withdrawal behaviors and an associated withdrawal-induced mechanical hypersensitivity. Brain slices prepared 24 h following the last morphine injection showed increases in ACC L5 thick-tufted PyN-intrinsic membrane excitability, increases in membrane resistance, reductions in the rheobase, and reductions in HCN channel-mediated currents (I-H). We did not observe changes in intrinsic or synaptic properties on thin-tufted, dopamine D1-receptor-expressing ACC L5 PyNs recorded from male Drd1a-tdTomato transgenic mice. In addition, we found that chemogenetic inhibition of the ACC blocked opioid-induced withdrawal and withdrawal-induced mechanical hypersensitivity. These results demonstrate that spontaneous opioid withdrawal alters neuronal properties within the ACC and that ACC activity is necessary to control behaviors associated with opioid withdrawal and withdrawal-induced mechanical hypersensitivity. The ability of the ACC to regulate both withdrawal behaviors and withdrawal-induced mechanical hypersensitivity suggests overlapping mechanisms between two seemingly distinguishable behaviors. This commonality potentially suggests that the ACC is a locus for multiple withdrawal symptoms.