Synaptic plasticity and functional improvement by dopamine transporter internalization after exposure to environmental enrichment

Abstract

Environmental enrichment (EE) with a complex combination of physical, cognitive and social stimulations enhances synaptic plasticity and behavioral function. However, the mechanism remains to be elucidated in detail. Therefore, this study attempted to investigate the underlying mechanisms associated with long-term exposure to EE by evaluating gene expression patterns. After then, this study validated the EE-mediated mechanism of synaptic plasticity. For this, six-week-old CD-1 mice were randomly assigned to either EE or control to investigate functional outcomes. EE mice were housed in a huge cage (86×76×31 cm3) for 2 mo, whereas control mice were housed in standard cages (27×22.5×14 cm3). Motor performances were evaluated using the rotarod test and ladder walking test and gene expression profile was investigated in the cerebral hemispheres using microarray and gene set enrichment analysis (GSEA). Subjects were recruited to evaluate striatal dopamine transporter (DAT) using a [18F]FPCIT-PET, surface DAT using biotinylation assay, DAT phosphorylation using proximity ligation assay (PLA) and immunoprecipitation (IP). In behavioral assessment, EE group showed significantly functional improvement in rotarod performance and ladder walking test. Microarray analysis revealed that genes associated with neuronal activity were significantly altered by EE. GSEA showed that genes involved in synaptic transmission and postsynaptic signal transduction were globally upregulated, whereas those associated with reuptake by presynaptic neurotransmitter transporters were downregulated. In particular, both microarray and GSEA demonstrated that EE exposure increased opioid signaling, acetylcholine release cycle, and postsynaptic neurotransmitter receptors but decreased Na+/Cl−-dependent neurotransmitter transporters, including DAT Slc6a3 in the brain. Since the striatum receives the largest dopamine input which involved motor performance in the brain, this study investigated that EE-induced functional improvement was related to DAT down-regulation in the striatum. In a [18F]FPCIT positron emission tomography scan, binding values of striatal DAT were significantly decreased approximately 18% in the EE mice relative to the control mice. DAT inhibitor administrated to establish the relationship of the DAT down-regulation to the treatment effects also improved rotarod performances, suggesting that DAT inhibition recapitulated EE-mediated treatment benefits. Next, EE-induced internalization of DAT was confirmed using a surface biotinylation assay. In situ proximity ligation assay and immunoprecipitation demonstrated that EE significantly increased the phosphorylation of striatal DAT as well as the levels of DAT bound with protein kinase C (PKC). In conclusion, EE enhanced motor function through the alteration of synaptic activity–regulating genes, improving the efficient use of neurotransmitters and synaptic plasticity by the downregulation of presynaptic reuptake by neurotransmitter transporters such as DAT. Therefore, this study suggests that EE enables phosphorylation of striatal DAT via a PKC-mediated pathway and causes DAT internalization.