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Purpose: To characterize newly discovered electrical synapses, formed by connexin (Cx) 36 and 45, between neighbouring axons within the optic nerve head. Methods: Twenty-five Wistar rats were killed by CO 2 inhalation. Proximal and distal optic nerve (ON) stumps were collected and processed for immunostainings, electron microscopy (EM) with immunogold labelling, PCR and Western blots (WB). Additional 15 animals were deeply anaesthetized, and flash visual evoked potentials (fVEP) after retrobulbar injection of saline (negative control) or 100 lM meclofenamic acid solution (gap junctions' blocker) were recorded. Human paraffin cross-sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research. Results: Immunostainings of both rat and human ON revealed the presence of Cx45 and 36 colocalizing with b3-tubulin, but not with glial fibrillary acidic protein (GFAP). In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 AE 0.005 in proximal stump and 0.44 AE 0.02 in distal stump), Cx45 showed higher levels (0.68 AE 0.01 in proximal stump and 0.9 AE 0.07 in distal stump). In immunogold-EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) directly coupling neighbouring axons. In fVEP, blocking of gap junctions with meclofenamic acid resulted in significant prolongation of the latency of P1 wave up to 160% after 30 min (p < 0.001). Conclusions: Optic nerve (ON) axons are equipped with electrical synapses composed of neuronal connexins, especially Cx45, creating direct morphological and functional connections between each other. This finding could have substantial implications for understanding of the pathogenesis of various optic neuropathies and identifies a new potential target for a therapeutic approach.Additional Supporting Information may be found in the online version of this article: Fig. S1. The method of VEP electrodes placement.

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PurposeTo evaluate impact of raised intraocular pressure on RNA binding protein ELAVL1/HuR expression in retina.MethodsIn order to induce glaucomatous damage, intraocular pressure was increased unilaterally using modified rat bead model and maintained for up to 8 weeks. Fellow eye was used as a healthy control. Animals were sacrificed, retina and optic nerves were collected and processed for western blot (WB) analysis or for immunostainings.ResultsGlaucomatous damage was confirmed in electron microscopy by the presence of axonal damage, myelin sheat dezintegration and glial cells proliferation within the optic nerve. Eight weeks glaucoma induced up to 36% loss of Retinal Ganglion Cells (RGCs) in the retina. In fractionated WB analysis of retinal homogenates, the level of active, cytoplasmic fraction of HuR was decreased approximately 3‐times when compared with healthy tissue (p < 0.05). Additionally, the cytoplasmic levels of cell cycle regulatory proteins (Hsp70, p53), whose expression is regulated by HuR, were also decreased. Stereological analysis of retinas revealed decrease in the number of double‐stained RGCs (positive for β3tubulin + HuR) below the number of total β3tubulin positive RGCs (1,960 ± 885 cells/mm2 vs. 2,136 ± 689 cells/mm2) what means that some of them lost visible HuR expression. Immunostaining of retinal and optic nerves cross sections showed decreased expression of HuR within RGCs and increased expression within optic nerve glia in glaucoma samples.ConclusionsIncreased intraocular pressure results in defect of active fraction of RNA binding HuR protein within retina and specifically within RGCs. This defect is linked with decreased expression of HuR‐dependent regulatory proteins (Hsp70, p53). This might be mechanism contributing to the development of glaucomatous degeneration.