Related Experiment Video
Updated: Jul 5, 2026
Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina
Published on: October 13, 2014
Glutamatergic calcium dynamics and deregulation of rat retinal ganglion cells
Andrew T E Hartwick1, Claire M Hamilton, William H Baldridge
1Retina and Optic Nerve Research Laboratory, Department of Anatomy & Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada.
Abstract:
A rise in intracellular calcium levels ([Ca(2+)](i)) is a key trigger for the lethal effects of the excitatory neurotransmitter glutamate in various central neurons, but a consensus has not been reached on the pathways that mediate glutamate-dependent increases of [Ca(2+)](i) in retinal ganglion cells (RGCs). Using Ca(2+) imaging techniques we demonstrated that, in the absence of external Mg(2+), the Ca(2+) signal evoked by glutamate was predominantly mediated by NMDA-type glutamate receptors (NMDA-Rs) in immunopanned RGCs isolated from neonatal or adult rats. Voltage-gated Ca(2+) channels and AMPA/kainate-Rs contributed a smaller portion of the Ca(2+) response at saturating concentrations of glutamate. Consistent with NMDA-R involvement, extracellular Mg(2+) inhibited RGC glutamate responses, while glycine had a potentiating effect. With Mg(2+) present externally, the effect of AMPA/kainate-R antagonists was enhanced and both NMDA- and AMPA/kainate-R antagonists greatly reduced the glutamate-induced increases of RGC [Ca(2+)](i). This finding indicates that the primary contribution of AMPA/kainate-Rs to RGC glutamatergic Ca(2+) dynamics is through the depolarization-dependent relief of the Mg(2+) block of NMDA-R channels. The effect of glutamate receptor antagonists on glutamatergic Ca(2+) signals from RGCs in adult rat retinal wholemounts yielded results similar to those obtained using immunopanned RGCs. Additional experiments on isolated RGCs revealed that during a 1 h glutamate (10-1000 microm) exposure, 18-28% of RGCs exhibited delayed Ca(2+) deregulation (DCD) and the RGCs that underwent DCD were positive for the death marker annexin V. RGCs with larger glutamate-evoked Ca(2+) signals were more likely to undergo DCD, and NMDA-R blockade significantly reduced the occurrence of DCD. Identifying the mechanisms underlying RGC excitotoxicity aids in our understanding of the pathophysiology of retinal ischaemia, and this work establishes a major role for NMDA-R-mediated increases in [Ca(2+)](i) in glutamate-related RGC death.
Insights
Glutamate excitotoxicity in retinal ganglion cells (RGCs) is primarily mediated by NMDA-type glutamate receptors (NMDA-Rs), leading to cell death. Blocking NMDA-Rs significantly reduces this glutamate-induced RGC death.
Area of Science:
- Neuroscience
- Ophthalmology
- Cell Biology
Background:
- Elevated intracellular calcium ([Ca(2+)](i)) triggered by glutamate causes neuronal death.
- Pathways of glutamate-induced [Ca(2+)](i) increases in retinal ganglion cells (RGCs) remain unclear.
- Understanding RGC excitotoxicity is crucial for retinal ischemia pathophysiology.
Purpose of the Study:
- To elucidate the mechanisms of glutamate-dependent [Ca(2+)](i) increases in RGCs.
- To determine the role of different glutamate receptors in RGC excitotoxicity.
- To investigate the contribution of NMDA-Rs to glutamate-induced RGC death.
Main Methods:
- Calcium (Ca(2+)) imaging techniques were employed on isolated RGCs and retinal wholemounts.
- Experiments utilized glutamate receptor antagonists (NMDA-R and AMPA/kainate-R), Mg(2+) manipulation, and glycine.
- Cell death was assessed using annexin V staining and delayed Ca(2+) deregulation (DCD) was observed.
Main Results:
- Glutamate-evoked Ca(2+) signals in RGCs were predominantly mediated by NMDA-type glutamate receptors (NMDA-Rs), especially without external Mg(2+).
- AMPA/kainate-Rs contributed to RGC Ca(2+) dynamics mainly by relieving the Mg(2+) block of NMDA-Rs.
- NMDA-R activation was linked to delayed Ca(2+) deregulation and annexin V-positive cell death in RGCs, with larger Ca(2+) signals increasing DCD likelihood.
Conclusions:
- NMDA-Rs play a major role in glutamate-induced increases of intracellular calcium in RGCs.
- NMDA-R-mediated calcium influx is a key pathway leading to glutamate excitotoxicity and RGC death.
- Blocking NMDA-Rs significantly reduces glutamate-induced RGC death, highlighting their therapeutic potential for retinal ischemia.
