Invulnerability of retinal ganglion cells to NMDA excitotoxicity

Abstract

NMDA excitotoxicity has been proposed to mediate the death of retinal ganglion cells (RGCs) in glaucoma and ischemia. Here, we reexamine the effects of glutamate and NMDA on rat RGCs in vitro and in situ. We show that highly purified RGCs express NR1 and NR2 receptor subunits by Western blotting and immunostaining, and functional NMDA receptor channels by whole-cell patch-clamp recording. Nevertheless, high concentrations of glutamate or NMDA failed to induce the death of purified RGCs, even after prolonged exposure for 24 h. RGCs co-cultured together with ephrins, astrocytes, or mixed retinal cells were similarly invulnerable to glutamate and NMDA, though their NMDA currents were 4-fold larger. In contrast, even a short exposure to glutamate or NMDA induced the rapid and profound excitotoxic death of most hippocampal neurons in culture. To determine whether RGCs in an intact retina are vulnerable to excitotoxicity, we retrogradely labeled RGCs in vivo using fluorogold and exposed acutely isolated intact retinas to high concentrations of glutamate or NMDA. This produced a substantial and rapid loss of amacrine cells; however, RGCs were not affected. Nonetheless, RGCs expressed NMDA currents in situ that were larger than those reported for amacrine cells. Interestingly, the NMDA receptors expressed by RGCs were extrasynaptically localized both in vitro and in situ. These results indicate that RGCs in vitro and in situ are relatively invulnerable to glutamate and NMDA excitotoxicity compared to amacrine cells, and indicate that important, as yet unidentified, determinants downstream of NMDA receptors control vulnerability to excitotoxicity.

Introduction

Although glutamate is the major excitatory neurotransmitter in the CNS, it is sometimes able to kill neurons Choi, 1992, Rothman and Olney, 1987. Excitotoxic neuronal death is thought to be an important contributor to neuron death caused by brain and spinal cord injuries as well as by many neurological diseases. An unsolved mystery, however, is why only some types of neurons are vulnerable to glutamate excitotoxicity. The ionotropic glutamate receptors are ligand-gated ion channels that are grouped into three pharmacologically defined classes: NMDA, AMPA, and kainate receptors. These receptors are encoded by at least six gene families: a single family for AMPA receptors (GluR1, 2, 3, 4), two for kainate (GluR5, 6, 7 and KA1, 2), and three for NMDA (NR1, NR2A, B, C, D, and NR3A, B) (Dingledine et al., 1999). These subunits combine into multimeric complexes to form functional receptor channels. To form an NMDA receptor, for instance, two NR1 subunits must combine with at least two NR2 subunits. NMDA receptors have been linked to excitotoxic death that occurs in less than 1 h of exposure, whereas much longer exposures to kainate or AMPA produce excitotoxicity (Choi, 1992).

We have focused on the effects of glutamate on retinal ganglion cells (RGCs) in the developing rat retina. RGC death is a leading cause of blindness resulting from glaucoma and retinal ischemia, and NMDA receptor-mediated excitotoxicity is presently thought to be an important contributor to RGC death in these conditions Ritch, 2000, Wax and Tezel, 2002. The expression of glutamate receptors by retinal ganglion cells in developing and adult rats has been extensively studied. Based on in situ hybridization and immunohistochemical studies of rat retinal sections, RGCs have been shown to express NMDA, AMPA, and kainate receptors including NR1, NR2A, B, C, D, GluR2, GluR7, and KA2 Brandstatter et al., 1994, Grunder et al., 2000a, Grunder et al., 2000b, Watanabe et al., 1994. Electrophysiological recordings of rat retinal ganglion cells in situ clearly indicate that they express functional NMDA and non-NMDA receptor-activated channels (Chen and Diamond, 2002). NMDA, AMPA, and kainate-activated currents can all be observed in RGCs in mixed retinal cultures over the 1-day period in which these cells remained viable without added neurotrophic support Aizenman et al., 1988, Taschenberger et al., 1995. In vitro and in vivo, rodent RGCs have been repetitively shown to be vulnerable to NMDA-mediated excitotoxicity (reviewed by Lipton, 2001, Sucher et al., 1997). For instance, both glutamate and NMDA induce the death of RGCs in postnatal and adult retinas Izumi et al., 1995, Li et al., 1999, Mosinger et al., 1991, Sabel et al., 1995. Although kainate-elicited excitotoxicity of RGCs in culture has not generally been observed, one group reported that kainate killed rat RGCs in purified cultures (Otori et al., 1998); however, kainate-induced excitotoxicity in intact retinas appears to be mediated by release of glutamate which then activates NMDA receptors Lipton, 2001, Sucher et al., 1997. Taken together, these studies indicate that the postnatal and adult RGCs express functional NMDA and non-NMDA receptors, and are vulnerable to NMDA-mediated excitotoxicity.

Because of their expected vulnerability to glutamate, we initially took measures to avoid exposing RGCs to glutamate in our studies involving their purification and culture (Barres et al., 1988). But eventually we were surprised to find that glutamate exposure actually enhanced the survival of purified RGCs in culture (Meyer-Franke et al., 1995). We found that depolarization enhanced RGC survival by enhancing neurotrophic responsiveness Goldberg and Barres, 2000, Goldberg et al., 2002, Meyer-Franke et al., 1998, Shen et al., 1999, but we remained puzzled by the invulnerability of the purified RGCs to excitotoxicity.

Here, we have investigated the glutamate and NMDA invulnerability of purified RGCs in vitro. By culturing highly purified RGCs in serum-free conditions, we have found that RGCs in vitro continue to express the same glutamate receptor subunits that they do in vivo, yet are invulnerable to glutamate and NMDA excitotoxicity. We also found that in vitro, as in situ (Chen and Diamond, 2002), NMDA receptors are localized extrasynaptically and not synaptically. To our surprise, we found that RGCs in intact developing and adult retinas are completely invulnerable to glutamate and NMDA excitotoxicity. Glutamate and NMDA did kill many cells in the inner nuclear layer (INL) and ganglion cell layer, but these cells were amacrine cells. Taken together, these studies cast doubt on the relevance of excitotoxicity to RGC death in glaucoma and retinal ischemia and strikingly illustrate that the presence of functional NMDA receptors is insufficient to account for excitotoxicity.