Assessment of Ca²⁺-permeable AMPA receptor calcium dynamics in ganglion cell layer neurons in an animal model of ocular hypertension

Abstract

Purpose: Glaucoma leads to loss of retinal ganglion cells (RGCs); however, the exact mechanisms leading to RGC death are not yet fully determined. One possible contributing mechanism is excessive influx of Ca²⁺ due to increased expression of Ca²⁺-permeable AMPA receptors (cpAMPARs). We examined this hypothesis by studying cpAMPAR-mediated Ca²⁺ dynamics in a rat model of ocular hypertension (OH).

Methods: OH was induced in 6 male Brown Norway rats by intraocular injection of polystyrene beads into the anterior chamber of one eye with the other eye serving as a control. Intraocular pressure (IOP) was measured with rebound tonometry. Retinas were harvested for imaging after 1-2 months of OH. AMPA-induced changes in ganglion cell layer neuron (GCN) intracellular calcium ([Ca²⁺]_i) was quantified by fura-2, loaded into rat GCNs by electroporation, in isolated whole retina. The influence of cpAMPARs was assessed by comparing AMPA-induced (50 µM) increases of [Ca²⁺]_i in the presence or absence of the cpAMPAR antagonist IEM-1460 (100 µM). To eliminate the confounding effects of NMDA receptors, all Ca²⁺-imaging experiments were performed in the presence of 20 µM MK-801. Following Ca²⁺ imaging the number of RGCs was subsequently assessed using immunohistochemistry for the RGC marker Brn3a.

Results: On average, OH rat eyes had a peak IOP increase (ΔIOP) of 16 ± 6 mm Hg (mean ± SD) and a 46% ± 19% loss of RGCs after 1-2 months compared to control (p<0.05, t-test). Calcium imaging of control eyes confirmed the presence of cpAMPARs in GCNs in that IEM-1460 decreased the AMPA-induced increase of fura-2 fluorescence to 47% (median, IQR 31%-70%; p<0.001, Friedman test and Dunn’s multiple comparison test; 111 cells from 5 retinas), an effect that was at least partially reversible (recovery to 75%, IQR 39%-99%) following 15 min wash. Similar results were found in GCNs from OH eyes: IEM-1460 decreased the AMPA-induced Ca²⁺ response to 48% (IQR 28%-79%, p<0.001, 110 cells from 6 retinas) with recovery to 66% (IQR 44%-100%) following wash.

Conclusions: The portion of the AMPA-induced [Ca²⁺]_i increase blocked by IEM-1460 was similar in GCNs from eyes subjected to OH as compared fellow control eyes. This result is not consistent with an increased expression of cpAMPARs in GCNs in ocular hypertensive eyes.

Keywords: 439 calcium • 531 ganglion cells • 517 excitatory amino acid receptors