ABSTRACT: Morphine is used to sedate critically ill infants to treat painful or stressful conditions associated with intensive care. Whether neonatal morphine exposure affects microRNA (miR) expression and thereby alters mRNA regulation is unknown. We tested the hypothesis that repeated morphine treatment in stress-exposed neonatal mice alters hippocampal mRNA and miR gene expression. C57BL/6 male mice were treated from postnatal day (P) 5 to P9 with morphine at 2 or 5 mg/kg ip bid (MS5) and then exposed to stress consisting of hypoxia (100% N2 1 min and 100% O2 5 min) followed by 2h maternal separation. Control mice were untreated and dam-reared. mRNA and microRNA expression profiling was performed on hippocampal tissues at P9. Overall, MS2 and MS5 morphine treatment altered expression of a total of 150 mRNAs (>1.5 fold change, P<0.05; 36 up, 114 down), and MS5 affected 63 mRNAs. The most upregulated mRNAs were fidgetin, arginine vasopressin, and resistin-like alpha, and the most down-regulated were defensin beta 11, aquaporin 1, calmodulin-like 4, chloride intracellular channel 6, and claudin 2. Gene Set Enrichment Analysis revealed that morphine treatment affected pathways related to cell cycle, membrane function, signaling, metabolism, cell death, transcriptional regulation, and immune response. MS5 decreased expression of miR-204-5p, miR-455-3p, miR-448-3p, and miR-574-3p.Nine morphine-responsive mRNAs that are involved in neurodevelopment, neurotransmission, and inflammation are predicted targets of the aforementioned differentially expressed microRNAs These data establish that morphine produces dose-dependent changes in both hippocampal mRNA and miR gene expression in stressed neonatal mice. If permanent, morphine–mediated neuroepigenetic effects may affect long-term hippocampal function, and this provides a mechanism for the neonatal morphine-related impairment of adult learning.
