A Master Regulator of Opioid Reward in the Ventral Prefrontal Cortex

Abstract Background Opioids that activate μ-opioid receptors (μORs) can elicit rewarding effects, contributing to opioid use disorder (OUD). Opioids can also trigger aversive reactions that protect against misuse. The cellular and molecular mechanisms in the brain that govern the interplay between opioid reward and aversion are poorly understood. Identifying the neural substrates that mediate opioid-induced aversion could provide insights into addiction vulnerability and withdrawal severity. Aims & Objectives This study aimed to identify the neural circuits and molecular mechanisms underlying hedonic reactions to opioids. First, we sought to identify brain regions in which neural activity was modified by a rewarding dose of oxycodone. Then, we sought to investigate role for opioid-responsive neurons in regulate opioid reward or aversion. Finally, we sought to identify the neurons that regulate opioid reward/aversion using high-resolution transcriptional profiling and determine their circuit-based mechanisms of action using single-cell connectomic mapping. Method FosTRAP2 mice and whole-brain c-Fos mapping was used to identify brain regions in which neural activity was modified by oxycodone. FosTRAP2 mice also enabled selective labeling and optogenetic manipulation oxycodone-regulated neurons. Multiplexed Analysis of Projections by Sequencing (MAPseq) was used to define the projection profiles of neurons. High-resolution spatial transcriptomics and single-nuclei RNA sequencing (snRNA-seq) characterized the molecular identity of relevant neurons. Whole-cell electrophysiology examined opioid modulation of neuronal activity. μORs were genetically ablated from neurons using μOR-floxed mice to assess their role in opioid reward and aversion. Results Whole-brain c-Fos mapping revealed oxycodone modified neural activity in the dorsal peduncular nucleus (DPn), located in the ventral prefrontal cortex. Optogenetic stimulation of DPn neurons produced an aversive state, which was reversed by oxycodone administration, indicating opioid suppression of DPn-mediated aversion. Using FosTRAP2 mice, we identified oxycodone-activated DPn neurons that project to the parabrachial nucleus (PBn). Optogenetic activation of DPn terminals in the PBn elicited an aversive response, which oxycodone blocked. Spatial transcriptomics identified a unique subpopulation of DPn pyramidal neurons expressing μORs and vesicular glutamate transporter 2 (vGlut2), genes not typically expressed by cortical glutamatergic neurons. Whole-cell electrophysiology confirmed that opioids inhibit vGlut2-expressing DPn neurons that project to the PBn. Selective deletion of μORs from DPn neurons rendered oxycodone aversive and intensified opioid withdrawal symptoms. Conversely, chemogenetic inhibition of DPn neurons alleviated withdrawal severity in oxycodone-dependent mice. Discussion & Conclusions This study identifies a previously unrecognized cortical circuit from the DPn to PBn that regulates opioid aversion and withdrawal. vGlut2-exprressing DPn neurons are unique among cortical excitatory neurons in their expression of μORs, allowing direct opioid inhibition. Activation of the DPn-PBn circuit drives opioid aversion, while opioid inhibition of these neurons suppresses this response. Genetic removal of μORs from DPn neurons shifts the hedonic balance of opioids toward aversion and exacerbates withdrawal symptoms. These findings establish the DPn as a critical hub in opioid-related behaviors and suggest that alterations in DPn function could influence susceptibility to OUD.