Single-cell transcriptomics reveals probiotic reversal of neonatal morphine-induced gene disruptions underlying adolescent pain hypersensitivity

Neonatal morphine is commonly administered in the Neonatal Intensive Care Unit (NICU) to manage pain. However, its long-term effects on neurodevelopment of pain pathways, remain a significant concern. The midbrain is a core region that plays a central role in pain processing and opioid-mediated analgesia. Here, we performed single-cell RNA sequencing to study gene expression in 107,427 midbrain single cells from adolescent mice neonatally exposed to either saline, morphine, or morphine with the probiotic Bifidobacterium infantis ( B. infantis ). We found broad alterations in transcriptomics within neurons, astrocytes, oligodendrocytes, and microglial cells. Analysis of differentially regulated genes revealed down regulation of HOX genes and upregulation of pathways related to neurotransmitter signaling and pain in adolescence that were neonatally treated with morphine. Interestingly, neonatal probiotic supplementation mitigated these morphine-induced alterations on the transcriptome. This study presents the first single-cell RNA sequencing dataset of the adolescent midbrain following neonatal morphine exposure and probiotic intervention. These findings offer new insights into the neurodevelopmental impact of early opioid exposure and highlight the therapeutic potential of microbiome-targeted interventions.