Electrical synapses interconnecting axons revealed in the optic nerve head - a novel model of gap junctions' involvement in optic nerve function

Abstract

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 CO₂ 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 μm 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 β3-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 ± 0.005 in proximal stump and 0.44 ± 0.02 in distal stump), Cx45 showed higher levels (0.68 ± 0.01 in proximal stump and 0.9 ± 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.

Keywords: conduction resistance; electrical synapses; impulse conduction; optic nerve; retinal ganglion cell axons.