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Maren Engelhardt, Annabelle Schlueter, Sabrina Rossberger, Silke Vorwald, Christian Schultz; Activity-dependent regulation of the cisternal organelle in the axon initial segment of retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1789.
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© ARVO (1962-2015); The Authors (2016-present)
The structural components that allow a neuron to function properly within its dynamic environment are organized in electrogenic microdomains such as the axon initial segment (AIS), the site of action potential generation. Cortical AIS exhibit striking plasticity in length and position along the axon during physiological and pathophysiological conditions. However, how the AIS of retinal ganglion cells (RGCs) is organized, if it exhibits similar plasticity, and which underlying mechanism drives this plasticity remains largely unknown. Here, we investigated the AIS-specific cisternal organelle (CO), a putative Ca2+-storage and release compartment, as a potential factor for AIS plasticity in RGCs.
Retinae of wildtype mice (n=6) at various developmental stages (P8-67) were perfusion-fixed and processed for whole mount immunofluorescence using antibodies against AIS scaffolding proteins ankyrinG, beta IV-spectrin and CO-marker synaptopodin. To address the dynamic regulation of the CO, confocal images were scanned at 600x and maximum intensity overlays were created from z-stacks. AIS length and CO cluster size analysis was performed with self-programmed ImageJ macros; Kruskal-Wallis tests were used for statistical analysis. To address the precise subcellular structure and localization of the CO in RGC axons, we applied Super-resolution Microscopy with a combination of 3D Structured Illumination Microscopy and Localization Microscopy.
Morphometrical analysis revealed that a subset of RGC AIS contain CO clusters (11% of all RGC). These show significant dynamic regulation during development in an activity-dependent manner, indicated by size increase until eye opening, followed by significant size reduction after light exposure (9.2µm2±1.05, P15; 6.3µm2±0.9, P21, p=0.001). A periodic distribution of scaffolding proteins extending from the axonal surface towards the inner cytoskeleton was evident within the AIS and a scaffold-deficient subdomain correlating with CO cluster location was identified.
Our data are consistent with the hypothesis that the CO of RGC AIS shows plasticity in an activity-dependent manner, observed as a dynamic structural remodeling of the CO during development. Further studies will now address how these structures and mechanisms are compromised during retinal disease.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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