Invulnerability of retinal ganglion cells to NMDA 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.
Section snippets
Expression of NMDA receptor proteins by purified RGCs in culture
As an initial survey of glutamate receptor subunit expression by RGCs in culture, we first examined mRNA extracted from purified RGCs after 3 days of culture by Affymetrix gene chip analysis and found that mRNAs for NR1, NR2, GluR2, and KA2 subunits, but not KA1 or other AMPA subunits, were expressed (data not shown) closely mirroring the previously determined RGC pattern of expression in vivo Brandstatter et al., 1994, Grunder et al., 2000a, Grunder et al., 2000b. We had previously reported
Purified RGCs express functional extrasynaptic NMDARs and NMDA current density is greatly enhanced by other cell types
RGCs in mixed retinal cultures have previously been shown to express NMDA currents Aizenman et al., 1988, Taschenberger et al., 1995, but we investigated whether highly purified RGCs in culture similarly expressed NMDA currents because of their invulnerability to NMDA excitotoxicity. The present findings show that purified RGCs express the NMDA receptor subunits NR1, NR2A, and NR2B, as measured by immunostaining and Western blotting, and that these subunits are assembled into functional NMDARs.
Cell purification and culture
Step-by-step protocols for all procedures are available on request from [email protected]. RGCs from postnatal days 5–21 (P5–P21) albino rats (Simonson rats; Simonson Labs, Gilroy, CA) were purified as previously described (Barres et al., 1988). Briefly, dissected retinas were enzymatically dissociated in papain in Dulbecco's phosphate-buffered saline; Gibco, Carlsbad, CA) to create a single-cell suspension. RGCs were isolated from this suspension using sequential immunopanning to greater
Acknowledgements
This work was supported by the National Eye Institute (R01 11030 to B.A.B.), and the March of Dimes Birth Defects Foundation (FY01-0503 to B.A.B.), a Zaffaroni Fellowship (E.M.U.), a HHMI undergraduate fellowship (W.B), and the NINDS Intramural Research Program (S.C and J.S.D.). We thank Regeneron for generously providing BDNF and CNTF.
References (98)
- et al.
Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxicity
Cell Calcium
(2003) - et al.
Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning
Neuron
(1988) - et al.
Influence of corticostriatal afferents on striatal kainic acid neurotoxicity
Neurosci. Lett.
(1978) Glutamate neurotoxicity in cortical cell culture is calcium dependent
Neurosci. Lett.
(1985)- et al.
EphB receptors interact with NMDA receptors and regulate excitatory synapse formation
Cell
(2000) - et al.
Lack of excitotoxic cell death in serum-free cultures of rat cerebral cortex
Brain Res.
(1990) - et al.
Retinal ganglion cells do not extend axons by default: promotion by neurotrophic signaling and electrical activity
Neuron
(2002) - et al.
An ex vivo rat retinal preparation for excitotoxicity studies
J. Neurosci. Methods
(1995) - et al.
Neuroprotective effects of BDNF in eyes with NMDA induced neuronal death
Brain Res.
(2000) - et al.
Hypoxic and excitotoxic damage to cultured rat retinal ganglion cells
Exp. Eye Res.
(1996)
Displaced amacrine cells in the ganglion cell layer of the hamster retina
Vision Res.
Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture
Neuron
Depolarization and cAMP elevation rapidly recruit TrkB to the plasma membrane of CNS neurons
Neuron
Ergothioneine treatment protects neurons against N-methyl-d-aspartate excitotoxicity in an in vivo rat retinal model
Neurosci. Lett.
Apoptotic cell death in the mouse retinal ganglion cell layer is induced in vivo by the excitatory amino acid homocysteine
Exp. Eye Res.
Blockade of both NMDA and non-NMDA receptors is required for optimal protection against ischemic neuronal degeneration in the in vivo adult mammalian retina
Exp. Neurol.
Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays
J. Immunol. Methods
New horizons in neuroprotection
Surv. Ophthalmol.
The role of group I and group II metabotropic glutamate receptors in modulation of striatal NMDA and quinolinic acid toxicity
Exp. Neurol.
Development of N-methyl-d-aspartate excitotoxicity in cultured hippocampal neurons
Brain Res. Dev. Brain Res.
Neuronal death in glaucoma
Prog. Retinal Eye Res.
The intact isolated (ex vivo) retina as a model system for the study of excitotoxicity
Prog. Retinal Eye Res.
Excitotoxicity and the NMDA receptor
Trends Neurosci.
Retinal ganglion cells lose trophic responsiveness after axotomy
Neuron
In situ localization of neuronal nitric oxide synthase (nNOS) mRNA in the rat retina
Neurosci. Lett.
Calcium channel antagonists attenuate NMDA receptor-mediated neurotoxicity of retinal ganglion cells in culture
Brain Res.
Molecular basis of glutamate toxicity in retinal ganglion cells
Vision Res.
Mobile NMDA receptors at hippocampal synapses
Neuron
Differential distributions of the NMDA receptor channel subunit mRNAs in the mouse retina
Brain Res.
The localization of nitric oxide synthase in the rat eye and related cranial ganglia
Neuroscience
Responses mediated by excitatory amino acid receptors in solitary retinal ganglion cells from rat
J. Physiol.
Resistance of retinal ganglion cells to an increase in intraocular pressure is immune-dependent
Invest. Ophthalmol. Visual Sci.
Culturing Nerve Cells
Proc. Natl. Acad. Sci. U. S. A.
Expression of NMDA and high affinity kainate receptor subunit mRNAs in the adult rat retina
Eur. J. Neurosci.
DCG-IV selectively attenuates rapidly triggered NMDA-induced neurotoxicity in cortical neurons
Eur. J. Neurosci.
Hyperthermia and hypoxia increase tolerance of retinal ganglion cells to anoxia and excitotoxicity
Invest. Ophthalmol. Visual Sci.
Synaptically released glutamate activates extrasynaptic NMDA receptors on cells in the ganglion cell layer of the rat retina
J. Neurosci.
Excitotoxic cell death
J. Neurobiol.
Glutamate neurotoxicity in cortical cell culture
J. Neurosci.
Activity-dependent recruitment of extrasynaptic NMDA receptor activation at an AMPA receptor-only synapse
J. Neurosci.
Private investigations
Nature
The glutamate receptor ion channels
Pharm. Rev.
Greater sensitivity of larger retinal ganglion cells to NMDA mediated cell death
Neuroreport
Elevated glutamate levels in the vitreous body of humans and monkeys with glaucoma
Arch. Ophthalmol.
Synaptic localization of NMDA receptor subunits in the rat retina
J. Comp. Neurol.
The relationship between neuronal survival and regeneration
Annu. Rev. Neurosci.
Distinct roles for ionotropic and metabotropic glutamate receptors in the maturation of excitatory synapses
J. Neurosci.
Distribution and developmental regulation of AMPA receptor subunit proteins in rat retina
Invest. Ophthalmol. Visual Sci.
Cited by (118)
Molecular mechanisms of ischemia and glutamate excitotoxicity
2023, Life SciencesNeuroprotection: A versatile approach to combat glaucoma
2020, European Journal of PharmacologyCitation Excerpt :Alternatively, the Mueller cells are affected by NMDA through a mechanism that is unidentified and secretes a neurotoxic TNF that promotes the death of RGC. Blocking glutamate receptors cannot improve the progression of the disease if chronic neurodegenerative disorders are containing an excitotoxic factor such as glaucoma without inhibiting certain harmful factors (e.g. TNF) (Ullian et al., 2004). The AMPA-type channels which are another form of glutamate receptor are the major contributor to neuronal injury.
- 1
These authors contributed equally to this paper.