The cerebellum has a well-established role in fine motor control and motor learning. In the cerebellar cortex, the first stage of information processing takes places at synapses between mossy fiber (MF) and cerebellar granule cell (GC) synapses, where synaptic transmission occurs at ultra-high frequencies (up to kHz). MF presynaptic machinery is capable of releasing glutamate at such extreme frequencies but the molecular determinants and ultrastructural organization underlying this presynaptic efficiency have yet to be elucidated. In several synapse types, the actin cytoskeleton, which is dynamically modulated by Rho GTPases, supports the supply of synaptic vesicles to active release sites during high-frequency stimulations. We hypothesize that Rho GTPases and actin filaments play a major role both in high-frequency glutamate release and in the morphological characteristics of MF-GC synapses. We first sought to obtain super-resolution 3D imaging of unitary MF to GC contacts. By combining multiplex imaging (virus-delivered click chemistry, immunohistochemistry) of sparse pre- and post-synaptic compartments and expansion microscopy, we achieved a characterization of unitary MF-GC connections at the nanoscale level. Further experiments are performed with dominant-positive or dominant-negative Rho-GTPases mutants specifically addressed to MF terminals to probe their involvement in both the synaptic ultrastructure and dynamic of glutamate release during high-frequency stimulation. This work provides important insight on the molecular determinants of high-frequency signaling.