Opioid receptor distribution in the claustrum-dorsal endopiriform complex

The claustrum and dorsal endopiriform regions form a thin subcortical sheet reciprocally connected to the neocortex that is enriched in opioid receptors. The precise cellular distribution of opioid receptors within this region remains unclear. Using multiplexed fluorescence in situ hybridization (mFISH) and hierarchical cluster analysis, we mapped the expression of mu (Oprm), delta (Oprd), kappa (Oprk), and ORL1 (Oprl) opioid receptor genes at single-cell resolution in the mouse claustrum-dorsal endopiriform. Among 2,269 neurons analyzed, six transcriptional clusters were identified: three excitatory (CLA, CLA/OPRK, cortical excitatory) and three inhibitory (SST, PVALB, broad inhibitory). Oprk expression was uniquely restricted to excitatory claustrum neurons and particularly enriched in the CLA/OPRK cluster which was comprised largely of Synpr+ claustrum core projection cells. In contrast, Oprd, Oprm, and Oprl genes were more broadly distributed across both excitatory and inhibitory populations, with Oprd enriched in parvalbumin (Pvalb) inhibitory neurons and Oprl in somatostatin (Sst) inhibitory neurons. Spatial mapping confirmed that Oprk expressing cells were concentrated within the claustrum core, whereas other receptor subtypes extended across the entire claustrum and into adjacent cortical regions. A comparable distribution of opioid receptors was observed in the neighbouring dorsal endopiriform. Analysis of a publicly available single cell sequencing dataset of the macaque claustrum also revealed a similar receptor distribution, where Oprk expression was enriched within a subset of excitatory projection neurons, and Oprm,Oprd, and Oprl expressed widely in both inhibitory and excitatory cells. Together, these findings demonstrate an evolutionarily conserved, cell-type-specific organization of opioid receptor expression in the claustrum-dorsal endopiriform. These results indicate that kappa opioid receptor expression is a defining molecular feature of excitatory claustrum projection neurons and suggest distinct roles for other opioid receptors in modulating inhibitory and cortical circuits.