The localization, regulation and function of synaptopodin and the cisternal organelle in the axon initial segment during murine visual system development

Activity-dependent development of neurons during critical periods of plasticity is a hallmark feature of visual system maturation. Synaptopodin (synpo) is an actin-associated protein and an essential component of the cisternal organelle (CO), localized in the axon initial segment (AIS). The CO is a putative Ca2+ store and potentially plays a role in Ca2+ current regulation of the AIS. The CO is the structural correlate of the spine apparatus in dendritic spines, which is a dynamic structure and exhibits striking plasticity depending on synpo expression. The AIS undergoes activity-dependent plasticity during visual cortex development, yet, how this plasticity is regulated remains to be elucidated. Furthermore, the structure of the AIS in retinal ganglion cells (RGCs) and its ability to undergo plasticity during retinal development is largely unknown. Therefore, the current thesis investigates the putative role of the CO in AIS structural plasticity in the murine visual system. For this purpose, the developmental regulation of synpo/CO expression in AIS was examined utilizing semi-quantitative immunofluorescence. Confocal microscopy and self-programmed macros were utilized to analyze synpo/CO cluster remodeling in AIS in the visual cortex and the retina. Activity-dependent regulation of synpo/CO expression was tested by performing visual deprivation experiments. The impact of synpo/CO expression on AIS maturation under normal and visual deprivation conditions was investigated in wildtype and synpo KO mice. The precise subcellular localization of synpo/CO in retinal AIS was determined applying super resolution microscopy. It was found that synpo/CO expression is dynamically regulated in an activity-dependent manner in a subset of visual cortex neurons and RGCs during visual system development. Dark-rearing of mice resulted in a significant increase in size and number of synpo/CO clusters in the AIS. Furthermore, a correlation between synpo/CO and structural AIS length maturation was found in the visual cortex. Visual deprivation led to the maintenance of elongated AIS with juvenile characteristics in adult wildtype, and to AIS shortening in synpo KO mice. This indicates a potential role for synpo/CO in AIS structural maturation. Super resolution analysis of retinal AIS revealed gaps in the AIS scaffold where synpo/CO clusters are located. In summary, the present thesis highlights that synpo/CO expression is regulated in an activity-dependent manner during visual system development and that the presence of synpo/CO influences structural AIS plasticity in the visual cortex