High-frequency oscillations (HFOs) have been observed to be particularly prominent in brain regions responsible for seizure initiation and propagation. Connexins play a crucial role in fast intercellular communication within the brain, as they are integral components of gap junctions, which facilitate direct cytoplasmic connections between neighboring cells. Recent studies identified clusters of connexins within the rodent CNS, specifically along the axon initial segment (AIS), a critical neuronal microdomain responsible for the initiation of action potentials. However, details about the subcellular organization of these clusters and their conservation across diverse brain regions and species are still to be uncovered. Our investigation revealed that connexin clusters are predominantly found in specific AIS microdomains where they align with intra-axonal calcium stores (cisternal organelle, CO). Through multi-channel immunofluorescence, confocal microscopy, and 3D reconstruction, we show that 86% of pyramidal neurons analyzed in primary motor cortex express connexin43 (Cx43) at the AIS, closely associated with synaptopodin, an actin-associated protein essential for CO formation. Multiple superresolution microscopy approaches such as 3D-STED, protein expansion microscopy and 3D-PAINT were employed to elucidate the structural relationships between synaptopodin-positive COs and axonal structures immunoreactive for Cx36, Cx43 and Cx45. Our ongoing research aims to confirm if the observed connexin clusters form bona fide gap junctions and to elucidate their role within the axonal microdomain. The importance of connexins in facilitating HFOs underscores their potential implications in epileptogenesis. Furthermore, the anticonvulsant effects observed with gap junction blockers indicate that these structures may be a promising target for new antiepileptic medications.