Abstract
Purpose: :
Glutamate receptors, including ionotropic and metabotropic receptors, mediate most excitatory neurotransmission in the retina and thus may contribute to the pathophysiology of retinal cell death. There are eight metabotropic glutamate receptor (mGluR) subtypes classified into three groups based on amino acid homology, agonist pharmacology and signal transduction mechanisms. We have demonstrated the expression of seven mGluRs in rat retinas and cultured retinal ganglion cells (RGCs). These include the group I (1, 5), group II (2, 3) and group III (4, 7, and 8) mGluRs, but not the mGluR6. We also examined the potential role of mGluR–mediated glutamate toxicity in RGC death by testing the effect of group–selective metabotropic glutamate agonists (mGluAs) on primary cultured RGCs.
Methods: :
RGCs were isolated from retinas of day–7 postnatal Sprague–Dawley rats using the Thy–1 immunopanning technique. The RGCs were seeded into 35mm dishes that were pre–plated with mixed primary rat retinal cells. These enriched RGC cultures were incubated for 5 days under normal conditions, then treated with 100µM of the group I mGluA, 3,5–dihydroxyphenylglycine (DHPG), the group II mGluA, 4–aminopyrrolidine–2,4–dicarboxylate (APDC), or the group III mGluA, L–2–amino–4–phosphonobutyric acid (L–AP4). After 24hrs, the cells were incubated with propidium iodide (PI) that labels the nuclei of dead cells, and were subsequently fixed and labeled with the selective RGC marker, Thy1. The number of PI–positive (dead) RGCs in each experimental group was counted (n>100) and compared to the control group. Statistical difference was assessed using one–way ANOVA.
Results: :
Immunocytochemistry studies show that seven of eight mGluR subtypes are expressed in cultured RGCs and in the RGC layer of retina. In addition, Thy–1 positive RGCs evaluated for cell death in response to the group–selective mGluAs revealed that activation of group I mGluRs, but not the group II and III mGluRs, leads to RGC death. Compared to control in which there was little PI staining, treatment with the group I mGluA DHPG induced a significant increase in RGC death (30%, p<0.01).
Conclusions: :
We have demonstrated that seven of eight mGluR subtypes are expressed in cultured primary RGCs and in retinal slice. In addition, activation of the group I mGluRs resulted in significant RGC death, while activation of group II and III mGluRs did not induce RGC death. These data suggest that group I mGluRs may contribute to glutamate–mediated RGC death and may involve in RGC pathophysiology.
Keywords: ganglion cells • receptors