Ocular hypertension increases expression of Ca²⁺-permeable AMPA receptors in α OFF RGCs

Abstract

Purpose :
We have previously shown that elevated hydrostatic pressure increases expression of Ca²⁺-permeable AMPA receptors (CP-AMPARs) in select populations of retinal ganglion cells (RGCs) in vitro. Here we test the hypothesis that ocular hypertension (OH) similarly increases CP-AMPARs in vivo.

Methods :
Eyes of C57Bl/J and KCNG4^cre;TdTomato mice were injected with microbeads. Intraocular pressure was measured once a week following bead injection. 2-3 weeks later, retinas were isolated and whole cell recordings of light responses from RGCs under dark adapted conditions (C57Bl/J), or responses to puffs of AMPA under light adapted conditions (Kcng4^cre;TdTomato), were obtained.

Results :
We focused primarily on Off α RGCs, as they are thought to be most susceptible to OH. CP-AMPAR expression was accessed by measuring light-evoked currents at positive and negative holding potentials with 500 µM spermine, a voltage dependent blocker of CP-AMPAR, in the pipet solution. From these measurements, a rectification index (RI) was calculated. 1.0 indicates no CP-AMPARs, and 0.0 indicates all CP-AMPARs. The RI of cells in control retinas was 0.87±0.06 (n=13). In bead treated eyes, the RI decreased dramatically to 0.45±.03 (n=10), a highly significant difference (p<0.0001, Wilcoxon rank sum test). Furthermore, light sensitivity was decreased approximately 5-fold (control: I_halfmax= 0.33 R*/rod/s, n=9; bead-treated: I_halfmax=1.70 R*/rod/s, n=7; p=0.003). Measurements of light-evoked currents in normal and OH eyes are confounded by the possibility of changes in synaptic transmission or phototransduction upstream of RGCs. We therefore additionally measured AMPA currents directly by application of AMPA. Cells were identified using the Kcng4cre;TdTomato line and by imaging of dendrites following filling with Alex488. In eyes with normal IOP, the RI was 0.95±0.07 (n=10). In eyes with OH, RI was 0.60±0.06 (n=12) (p=0.003). Thus, both approaches demonstrate an increase in CP-AMPAR expression in Off a RGCs of hypertensive eyes.

Conclusions :
Our findings suggest that as little as a 25% increase in IOP for two weeks is sufficient to dramatically increase CP-AMPAR expression and decrease light sensitivity in Off α RGCs. An increase in Ca²⁺ influx may contribute to RGC loss in glaucomatous eyes. Further experiments will determine if a decrease in light sensitivity is related to CP-AMPAR or an independent consequence of elevated pressure.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.