Elevated ambient pressure modulates AMPA receptor expression in retinal ganglion cells.

Abstract

Purpose : To test the hypothesis that elevated hydrostatic pressure induces an increase in Ca²⁺-permeable AMPA receptors (CP-AMPARs) in an in vitro model of glaucoma.

Methods : Retinal ganglion cells (RGCs) from newborn mice were maintained in a mixed culture for up to 28 days. At 7-10 DIV, cultures were subjected to a hydrostatic pressure of 45-50 mm Hg for 14-18 hours by placing them in a custom designed chamber in a humidified atmosphere at 37 ° C and 5% CO₂. Functional measurements of CP-AMPAR surface expression were obtained by evoking AMPA currents in the absence and presence of CP-AMPAR blockers. To confirm that RGCs were consistently targeted for recording, a subset of cells were filled with neurobiotin, fixed and labeled with TUJ 1, an antibody that recognizes beta III tubulin, a reliable marker of RGCs. Both TUJ 1 and SMI-32, which labels putative α RGCs were used to visualize RGCs for morphological analysis.

Results : We elicited AMPA currents in the absence and presence of 100 µM PhTX 74, which blocks AMPARs that contain the GluA1,3 or 4 subunits. In control cells, a minority of the total AMPA current was conducted by CP-AMPARs (27.7 ± 3.0%; n=22 cells). When measured between 1-5 days after pressure treatment, cells from sister cultures increased expression of CP-AMPARs, resulting in increased conduction of AMPA current through CP-AMPARs (50.0 ± 3.3%; n=17 cells), a highly significant difference (p<0.0001; unpaired t-test). spontaneous EPSCs (sEPSCs) could often be observed in RGCs. To determine the relative proportion of CP-AMPARs at postsynaptic sites, we blocked sEPSCs with PhTX 74. In control cells, PhTX reduced the mean amplitude of sEPSCs by 57.4 ± 6.7%. The spontaneous frequency was also reduced, most likely due to a reduction in excitatory tone of RGCs providing synaptic input to the recorded cell. The effect of PhTX 74 was more dramatic in pressure treated RGCs, blocking sEPSCs almost completely. This is consistent with the hypothesis that elevated pressure drives insertion of CP-AMPARs into synapses. TuJ 1 and SMI-32-labeled RGCs revealed an increase in membrane blebbing 1-5 days after treatment with pressure. After 5 days, the number of RGCs was reduced by 32.5 ± 13% relative to control cultures.

Conclusions : Elevation of hydrostatic pressure to moderate levels for 14-18 hours is sufficient to promote an increase in synaptic and nonsynaptic CP-AMPARs, and to induce apoptosis in RGCs in vitro.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.