Single-Cell Sequencing Analysis Reveals Dysregulated Energy Metabolism and Altered Synaptic Connectivity in the Mouse Retina Following Dark Rearing.

Purpose: In this study, single-cell RNA sequencing (scRNA-seq) was applied to retinas from dark-reared and normally reared mice to resolve how each cell type contributes to retinal alterations under visual deprivation. Methods: C57BL/6J mice were reared in complete darkness from birth to postnatal day 36 (P36), when visual function and retinal morphology were evaluated. At P36, retinas were isolated for scRNA-seq, followed by transcriptomic profiling, cell clustering, differential expression, and cell-cell communication analyses. Key findings were validated by immunofluorescence, qPCR, Seahorse metabolic assays, flow cytometry, and targeted biochemical measurements. To determine the functional significance of Müller glia-derived DIO2, pharmacological inhibition was performed using iopanoic acid, followed by exogenous T3 supplementation. Results: Dark rearing markedly reduced visual function, as shown by attenuated optomotor responses and decreased electroretinogram a- and b-wave amplitudes. Photoreceptors exhibited downregulation of mitochondrial genes, accompanied by decreased oxygen consumption rate, reduced mitochondrial membrane potential, lower ATP levels, and progressive upregulation of Hif1α, collectively indicating impaired energy metabolism under visual deprivation. At the synaptic level, the presynaptic vesicle-priming protein UNC13A (Munc13-1) was selectively downregulated in ON-bipolar cells, whereas amacrine cells showed enhanced GABAergic signaling. Notably, dark rearing induced both systemic and local thyroid hormone fluctuations, triggering a dynamic compensatory upregulation of DIO2 specifically in Müller glia. Inhibiting DIO2 exacerbated visual dysfunction and retinal thinning, whereas T3 supplementation successfully rescued these deficits. Conclusions: This study reveals that dark rearing elicits metabolic dysregulation and synaptic remodeling in the retina. Müller glia-derived DIO2 acts as a critical compensatory mechanism to stabilize local thyroid hormone homeostasis, which is essential for preserving retinal structure and function.