Background:Opioids are efficacious and safe analgesic drugs in short-term use for acute pain but chronic use can lead to tolerance and dependence. Opioid-induced microglial activation may contribute to the development of tolerance and this process may differ between males and females. A link is suggested between this microglial activation and inflammation, disturbances of circadian rhythms, and neurotoxic effects. We set out to further delineate the effects of chronic morphine on pain behaviour, microglial and neuronal staining, and the transcriptome of spinal microglia, to better understand the role of microglia in the consequences of long-term high-dose opioid administration.Experimental Approach:In two experiments, we administered increasing subcutaneous doses of morphine hydrochloride or saline to male and female rats. Thermal nociception was assessed with the tail flick and hot plate tests. In Experiment I, spinal cord (SC) samples were prepared for immunohistochemical staining for microglial and neuronal markers. In Experiment II, the transcriptome of microglia from the lumbar SC was analysed.Key Results:Female and male rats had similar antinociceptive responses to morphine and developed similar antinociceptive tolerance to thermal stimuli following chronic increasing high doses of s.c. morphine. The area of microglial IBA1-staining in SC decreased after 2 weeks of morphine administration in both sexes. Following morphine treatment, the differentially expressed genes identified in the microglial transcriptome included ones related to the circadian rhythm,apoptosis, and immune system processes.Conclusions:Female and male rats showed similar pain behaviour following chronic high doses of morphine. This was associated with decreased staining of spinal microglia, suggesting either decreased activation or apoptosis. High-dose morphine administration also associated with several changes in gene expression in SC microglia, e.g., those related to the circadian rhythm (Per2, Per3, Dbp). These changes should be considered in the clinical consequences of long-term high-dose administration of opioids.