Effects of morphine self-administration on brain structure and microglial phenotypic diversity in the absence of neuronal loss in male Wistar rats

RATIONALE: Opioid addiction, including morphine use, is a major public health crisis in the U.S. It has been associated with brain volume changes in reward-related regions, neuronal loss, and neuroinflammation. While these alterations have been studied separately, it remains unclear whether structural changes co-occur with microglial adaptations at early stages of morphine use. OBJECTIVE: This study aimed to examine region-specific brain volume changes, cellular counts, and the emergence of distinct microglial phenotypes in addiction-related regions, using a model of morphine self-administration that simulates the early phase of morphine consumption. METHODS: Male Wistar rats were trained to self-administer morphine (0.01 mg/kg/inf) for 20 days in 3-hour daily sessions under operant conditioning. Structural MRI was conducted before and after the self-administration period, and brain volume was quantified using deformation-based morphometry. Brain tissue was immunolabeled for Iba1 and NeuN, and confocal microscopy images of microglia were analyzed using principal component analysis and K-means clustering. RESULTS: Morphine self-administration produced volume increases in the globus pallidus and reductions in the insular cortex. Microglial density was elevated in these regions and other addiction-related areas, including the caudate-putamen and dentate gyrus, without significant variations in neuronal count but with a marked reduction in neuronal soma size in these latter regions. Clustering revealed diverse microglial phenotypes, including intermediate morphologies, with region-dependent distributions indicative of diverse neuroinflammatory states. CONCLUSIONS: These findings suggest that morphine-induced brain volume changes during the early stages of consumption are not attributable to neuronal loss but may reflect adaptive processes involving neuronal restructuring and microglial remodeling. Microglial phenotyping emerges as a sensitive approach for detecting neuroinflammatory patterns linked to addiction vulnerability.