Mapping Nociceptive and Opioidergic Cell-Types in the Cingulate Cortex of Mice and Non-Human Primates

The anterior cingulate cortex (ACC) has emerged as a brain region of focus toward understanding the complex interplay between pain and opioid use disorders. However, little is known about the anatomical location and molecular identity of ACC nociceptive cell-types expressing the mu-opioid receptor (MOR)—the molecular target of opioid analgesics—and if this cortical architecture is conserved across species. Using single nuclei RNA sequencing of murine ACC, we found that three cell-subtypes of Slc17a7 glutamatergic neurons were the most transcriptionally active to noxious stimuli (identified by weighted expression of immediate early genes (IEGs) and differential gene expression analysis between nuclei from uninjured and chronic neuropathic pain mice). These cell-subtypes all co-expressed Oprm1 and single genetic identifiers, which we spatially resolved to label layer-specific, distinct neurons in L2, L5a, and L6b neurons. Next, we used immunohistochemistry and in situ hybridization to conduct layer-specific analyses of MOR+ and noxious IEG-responsive cells in mouse and rhesus macaque ACC. In both species, we found MOR expression distributed across all ACC layers but enriched in Layer 6a/6b Vglut1+ excitatory neurons, and that nociceptive cells are prominently localized to unique ACC sub-regions. Lastly, we report on a novel viral approach to gain genetic access within MOR+ cell-types in mouse and macaque ACC and validate the specificity of viral transgene expression against endogenous MOR expression. Together, our layer-specific analysis and new opioid-promoter viral tool present a targeted way to selectively engage these same cortical populations across translational models of pain and opioid use disorder for future neuromodulation studies.