Ca²⁺ signaling in retinal ganglion cells and their axons

Abstract

Purpose: Blocking voltage gated Ca channels (VGCCs) reduces degeneration of retinal ganglion cells (RGCs) caused by optic nerve trauma (Karim et al., 2006). RGC somata have been shown to express L-, N- and T-type VGCCs (Guenther et al., 1994; Farrell et al., 2009). Since axons are the primary site of nerve injury, we sought to compare the localization of VGCCs in RGCs and RGC axons to determine possible differences in their contribution to Ca²⁺ signaling.

Methods: Immunohistochemistry was performed on wholemount retinas and transverse sections. Ca²⁺ imaging was performed on retinas that were treated with specific blockers of L-, N-, P/Q-, and T-type Ca channels, nifedipine and verapamil, ω-conotoxin-GVIA, ω-Agatoxin IVA, and mibefradil, respectively. The Na channel blocker, TTX, was also used. RGC axons were labeled retrogradely with Fluo-4 pentapotassium injected at the optic nerve stump and depolarized by superfusion with 60 mM K⁺ for 30 or 60s.

Results: Immunolabeling showed N- and P/Q-type Ca channels in RGC somata. The α1C & D subunits were localized to RGC somata, the nerve fiber layer (NFL) and processes in the inner plexiform layer. Ca²⁺ imaging of RGCs in isolated retinas showed the following significant reductions in the K⁺-evoked Ca²⁺ signal: 10 μM nifedipine (32%±9%), 100 μM verapamil (39%±4%), 3 μM mibefradil (21%±5%) and 200 nM TTX (40%±9%). Ca²⁺ imaging of RGC axons in retinas showed the following significant reductions: nifedipine (20%±6%), verapamil (52%±9%), mibefradil (13%±8%) and TTX (48%±11%). 400 nM ω-Agatoxin IVA and 3 μM ω-conotoxin-GVIA tended to reduce the Ca²⁺ signal in RGC somata & axons but by an amount that failed to reach significance in this data set.

Conclusions: These findings suggest that much of the Ca²⁺ influx in both RGC somata and axons is due to L-type VGCCs and that spiking is a strong stimulus for Ca²⁺ influx. The expression of VGCC subtypes in RGCs and the selective reduction of Ca²⁺ signals in the presence of specific antagonists may provide insight into the development of target-specific strategies to spare the loss of RGCs and their axons following injury.

Keywords: 531 ganglion cells • 439 calcium • 449 cell survival