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Activity-dependent synapse plasticity is important for the establishment of neuron wiring in the central nervous system, particularly in the context of sensory processing. In the visual system, image-forming and non-image-forming retinal input into the brain is a popular model for studying activity-dependent plasticity due to the well-characterized neural activity and bulk-level innervation pattern. However, investigation of synaptic connection during early development has been impeded by the limited resolution of conventional fluorescent microscopy or lack of profile tagging in electron microscopy (EM) images. To overcome these challenges, we employed volumetric STochastic Optical Reconstruction Microscopy, immunohistochemistry synaptic protein labeling, and anterograde retinal tract tracing to investigate the activity-dependent retinogeniculate and retinohypothalamic synapse plasticity. Through our findings, we uncover the developmental pattern of retinofugal innervation and shed light on the impact of spontaneous activity on retinal synapse maturation at the synaptic level. During the first postnatal week in mice, the dorsal lateral geniculate nucleus (dLGN) initially receives overlapping input from the two eyes before the binocular innervation segregated. The changes in individual synapse properties during the eye-specific segregation process have remained unknown. In Chapter 2, we uncovered eye-specific differences in presynaptic vesicle pool size and vesicle association with the active zone at the earliest stages of retinogeniculate refinement but found no evidence of eye-specific differences in subsynaptic domain number, size, or transsynaptic alignment across development. Genetic disruption of spontaneous retinal activity decreased retinogeniculate synapse density, delayed the emergence of eye-specific differences in vesicle organization, and disrupted subsynaptic domain maturation. These results suggest that activity-dependent eye-specific presynaptic maturation underlies synaptic competition in the mammalian visual system. The dLGN relays visual information from the retina to the visual cortex through parallel processing pathways. In adult mice, such processing is achieved through spatial clustering of several retinal ganglion cells (RGCs) boutons to integrate convergent or divergent visual information. It is unknown whether such RGC synapse clustering occurs during the early developmental stage. In Chapter 3, we identified a subset of complex retinogeniculate synapses with larger presynaptic vesicle pools and multiple AZs that simultaneously promote the clustering of like-eye synapses (synaptic stabilization) and prevent synapse formation from the opposite eye (synaptic punishment). In mutant mice with disrupted spontaneous retinal wave activity, complex synapses are formed but fail to drive eye-specific synaptic clustering and punishment. These results reveal the early formation of a unique synaptic subset that regulates activity-dependent eye-specific synaptic competition and may serve as substrates for later synapse clustering formation. A subset of RGCs that express the photopigment melanopsin (OPN4) innervate the suprachiasmatic nucleus (SCN), which serves as the central pacemaker responsible for controlling circadian rhythm in mammals. The function of OPN4 is important for SCN photoentrainment, but its impact on retinal synapse maturation during early development is unknown. In Chapter 4, we found that OPN4 plays an important role in retinal synapse formation and activation in the SCN during the early developmental stage. Loss of OPN4 leads to reduced retinal synapse density, and increased variability in the ratio of synapses with few or no docking vesicles, but has not effect on total vesicle pool volume. Meanwhile, the subsequent maturation of retinohypothalamic tract (RHT) synapses after the first postnatal week shows diminished reliance on OPN4 function and further compensates for the early defects in the absence of OPN4. This study reveals a moderate influence of OPN4 on early RHT synapse development and sheds light on the role of photopigment in regulating SCN synapse plasticity. This dissertation introduces a novel approach using super-resolution fluorescent imaging in the thalamus and hypothalamus tissue. Our work has yielded insights into the activity-dependent maturation in synapse properties and spatial distribution in the dLGN, as well as the impact of OPN4 on retinohypothalamic synapses in the SCN. By revealing the synapse development at the molecular level, our study demonstrates presynaptic mechanisms that underlie activity-dependent retinal synapse plasticity during the early developmental stage. Furthermore, our application of super-resolution fluorescent microscopy highlights its potential as a valuable tool for future in situ studies on brain development.