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Adrian Smedowski, Saeed Akhtar, Lucia Podracka, Xiaonan Liu, Elisa Toropainen, Marialaura Amadio, Marita Pietrucha-Dutczak, Markku Varjosalo, Arto Urtti, Kai Kaarniranta, Joanna Lewin-Kowalik; Synaptic coupling of unmyelinated optic nerve axons – a novel insight into optic nerve structure and conductivity properties. Invest. Ophthalmol. Vis. Sci. 2018;59(9):313. doi: https://doi.org/.
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Axons in the optic nerve are arranged in bundles and conducting action potential with resistance related to their membrane. Gap junctions, formed by transmembrane proteins (connexins, cx), have been described to connect cytoplasm of optic nerve axons and astrocytes allowing intercellular passage of small molecules and ions. Here, we aim to characterize the function of newly identified gap junctions coupling optic nerve axons with each others.
Twenty male 8-week old Wistar rats were sacrificed by CO2 inhalation. Proximal and distal optic nerve stumps were collected and processed for immunostainings, electron microscopy (EM) with immunogold labeling, RT-PCR and western blots (WB). Additional 20 animals were deeply anesthetized and stainless-steel screw electrodes were drilled in skulls in standard locations to record fVEPs (Fig. 1A). Recordings were performed in untouched animals, after retrobulbar injection of 50 μl of saline (negative control) or 50 μl of 100 μM meclofenamic acid solution (gap junctions’ blocker). Human paraffin cross-sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research, University of Toronto, Canada.
Imunostaining of both rat and human samples revealed presence of Cx45 and 36 in axonal membranes, colocalizing with β3-tubulin, but not with GFAP. In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58±0.005 in proximal stump and 0.44±0.02 in distal stump), Cx45 showed higher levels (0.68±0.0003 in proximal stump and 0.9±0.07 in distal stump). Additionally, we detected transcript of Cx45 and Cx36 in RT-PCR analysis of optic nerve homogenates. In immunogold-EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) that are directly coupling neighboring axons. In fVEPs, blocking of gap junctions’ with meclofenamic acid resulted in significant prolongation of the latency of P1 wave up to 140% after 5 min from retrobulbar injection of this blocker (p<0.001) and 170% after 30 minutes (p<0.001).
Optic nerve axons do not form absolutely independent conductive channels. They are directly coupled by gap junctions formed in majority by neuronal Cx45. Coupling of axons, except known transpassing functions, allows to reduce axonal membrane resistance of optic nerve and accelerates transduction of visual signal (Fig. 1 B).
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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