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Striatal medium spiny neurons (MSNs) control motor function. Hyper- or hypo-activity of MSNs coincides with basal ganglia-related movement disorders. Based on the assumption that lasting alterations in neuronal activity lead to structural changes in the brain, understanding these structural alterations may be used to infer MSN functional abnormalities. To infer MSN function from structural data, understanding how long-lasting alterations in MSN activity affect brain morphology is essential. To address this, we utilized a simplified model of functional induction by stimulating MSNs expressing channelrhodopsin 2 (ChR2). Subsequent structural alterations which induced long-term activity changes in these MSNs were investigated in the striatal pathway and its associated regions by diffusion tensor imaging (DTI) and histological assessment with super-resolution microscopy. DTI detected changes in the striatum, substantia nigra, and motor cortex. Histological assessment found a reduction in the diameter of myelinated cortical axons as well as MSN dendrites and axons. The structural changes showed a high correlation between DTI parameters and histological data. These results demonstrated that long-term neural activation in the MSNs alters the diameter of MSN and cortical neurons fibers. This study provides a tool for understanding the causal relationship between functional and structural alterations.Long-term optogenetic stimulation in MSNs induced motor dysfunction.DTI found changes in DTI parameters in the striatal pathway.Super-resolution microscopy confirmed structural changes described by DTI.Long activation in MSNs alters the diameter of MSN fibers and cortical fibers.