Regulation of Excitatory Amino Acid Transmission in the Retina: Studies on Neuroprotection

Abstract

Excitotoxicity occurs in neurons due to the accumulation of excitatory amino acids such as glutamate in the synaptic and extrasynaptic locations. In the retina, excessive glutamate concentrations trigger a neurotoxic cascade involving several mechanisms, including the elevation of intracellular calcium (Ca²⁺⁾ and the activation of α-amino-3-hydroxy 5-methyl-4-iso-xazole-propionic acid/kainate (AMPA/KA) and N-methyl-d-aspartate (NMDA) receptors leading to retinal degeneration. Both ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) are present in the mammalian retina. Indeed, due to the abundant expression of GluRs, the mammalian retina is highly susceptible to excitotoxic neurodegeneration. Excitotoxicity has been postulated to present a common downstream mechanism for several stimuli, including hypoglycemia, hypoxia, ischemia, and chronic neurodegenerative diseases. Experimental approaches to the study of neuroprotection in the retina have utilized insults that trigger hypoxia, hypoglycemia, or excitotoxicity. Using these experimental approaches, the neuroprotective potential of GluR agents, including the NMDA receptor modulators (MK801, ifenprodil, memantine); AMPA/KA receptor antagonist (CNQX); Group II and III mGluR agonists (LY354740, quisqualate); and Ca²⁺-channel blockers (diltiazem, lomerizine, verapamil, ω-conotoxin), and others (pituitary adenylate cyclase activating polypeptide, neuropeptide Y, acetylcholine receptor agonists) have been elucidated. In addition to corroborating the exocytotic role of excitatory amino acids in retinal degeneration, these studies affirm that multiple mechanism/s contribute to the prevention of damage caused by excitotoxicity in the retina. Therefore, it is feasible that several pathways are involved in protecting the retina from toxic insults in ocular neurodegenerative conditions such as glaucoma and retinal ischemia. Furthermore, these experimental models are viable tools for evaluating therapeutic candidates in ocular neuropathies.

Introduction

Laboratory research on neuroprotection in the retina have largely focused on glutamate, a major excitatory neurotransmitter in the retina that also plays an important role in the transmission of visual information into the central nervous system (CNS).^1,2 Upon its release from presynaptic terminals, glutamate activates receptors that are broadly classified into the ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs).² The iGluRs are classified into different subtypes based upon their selectivity as agonists into α-amino-3-hydroxy 5-methyl-4-iso-xazole-propionic acid (AMPA), kainate (KA), and N-methyl-d-aspartate (NMDA) subtypes with detailed nomenclature for each subunit.³ Among the iGluR, AMPA and KA are also known as non-NMDA subtypes and are permeable to Na⁺ and K⁺ ions, whereas the NMDA receptors are voltage-dependent channels with a relatively high permeability to Ca²⁺ ions.^2,4 The mGluRs are G-protein-coupled receptors with 7 transmembrane spanning domains displaying a binding site for glutamate on the extracellular N-terminal domain. Three groups of mGluRs consisting of eight receptor subtypes have been described: Group I (mGluR 1 and mGluR5), which activates G protein coupled to phospholipase C; Group II (mGluR 2–4); and Group III (mGluR 6–7) that inhibits adenylyl cyclase.² Since glutamate receptors have been implicated in excitotoxicity, a fundamental, common downstream mechanism associated with excessive glutamate-, hypoglycemia-, and hypoxia-induced excitotoxicity models, the present review article will provide an overview of role of glutamate metabolism in retinal neuroprotection both in vivo and in vitro.

Glutamate receptors in retina

Immunocytochemical studies reveal prominent glutamate immunoreactivity in photoreceptors, horizontal, bipolar, and ganglion cells of the retina.⁵ Indeed, glutamate has been reported to serve as the primary excitatory amino acid transmitter in these neurons.^6–8 As elsewhere in the CNS, glutamate's actions are terminated by its rapid uptake by Muller glial cells and/or presynaptic terminals. As such, if the latter uptake mechanisms become defective or are swamped by excess extracellular glutamate, there is a strong possibility that the high concentration of this excitotoxic amino acid will begin to overstimulate neurons in its vicinity and cause their demise. In support of this notion, both iGluRs and mGluRs have been identified in the mammalian retina,^9–12 and indeed many subtypes of retinal ganglion cells (RGCs) are highly susceptible to excitotoxicity due to the abundance of iGluRs on their cell surface.¹³

Ionotropic Glutamate Receptors

NMDA receptors

Structurally, the NMDA receptor is a heterotetramer consisting of 2 GluN1 (or NR1) and 2 GluN2 (or NR2) subunits.^14–17 Eight variants of the NR1 subunit (generated by alternative splicing of GRIN1), consisting of NR1-1a,1b to NR1-4a,4b have been identified.¹⁸ In vertebrates, 4 isoforms of the NR2 subunit, NR2A through NR2D (encoded by GRIN2A, GRIN2B, GRIN2C, GRIN2D) are expressed.¹⁸ With exception of NR2D, all the NMDA receptor subunits were detected in adult rat retina by in situ hybridization techniques.¹³ NR2A was localized in the inner plexiform layer of the rat, rabbit, and monkey retina.¹⁹ NR2A showed immunoreactivity in amacrine cell or ganglion cell process postsynaptic at cone bipolar cell ribbon synapses, while NR1 was detected in rod bipolar cells in mouse retina.²⁰ NR2D immunoreactivity has been detected in rod bipolar cells of rat and rabbit retina.²¹

The NMDA receptors exhibit agonist and antagonist selectivity, calcium permeability, magnesium blockade, and glycine modulation.^22–24 Activation of the NMDA receptor by glutamate or any other agonist elicits postsynaptic calcium influx and consequent increase in intracellular calcium. The elevation of intracellular calcium then activates various signaling cascades, including nitric oxide pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, and phosphorylation of Ca²⁺-cAMP response element binding protein (CREB).^25–28 Studies done on retinal ribbon synapses showed that extended NMDA receptor activation results in reciprocal inhibition through GABA_A and GABA_C receptors.²⁹ Furthermore, excessive activation of these receptors is associated with neuronal excitotoxicity.^30–33

AMPA receptors

AMPA receptors are tetrameric ion channels formed from GluR-A (GluR1), GluR-B (GluR2), GluR-C (GluR3), and GluR-D (GluR4) subunits. The receptors are composed of either homotetramers of GluR1 or GluR4 or symmetric dimers of GluR2/3 and either GluR1 or GluR4.³⁴ Using in situ hybridization studies, AMPA receptor subunits were localized in the inner nuclear layer and ganglion cell layer of mouse, rat, and cat retina.^35–37 GluR1–GluR4 subunits were localized in neurons in the retina of mouse, rat, and cat and confirmed by immunocytochemistry in horizontal, amacrine, ganglion, and cone bipolar cells in the cat retina.³⁸

Activation of AMPA receptors facilitates a rapid influx of Na⁺, K⁺, or Ca²⁺ ions depending on the subunits, followed by rapid receptor desensitization.^39–41 AMPA receptors that possess GluR2 subunit tend to be impermeable to Ca²⁺.⁴² Xia et al. reported that replacement of AMPA receptors that were impermeable to Ca²⁺ with Ca²⁺-permeable receptors lacking GluR2 caused an increase in Ca²⁺ influx in darkness.⁴³ Moreover, the increase in calcium associated with this response could activate CREB through Ca²⁺/calmodulin-dependent protein kinases.⁴⁴ It is conceivable that these GluR2-AMPA receptors serve a protective role against neuronal excitotoxicity.⁴⁵

KA receptors

KA receptors are assembled from 5 subunits, namely GluR5 (GRIK1), GluR6 (GRIK2), GluR7 (GRIK3), KA1 (GRIK4), and KA2 (GRIK5), to form heteromeric channels that can be desensitized with glutamate and KA. The GluR5, GluR6, and GluR7 subunits have low affinity for KA, while KA1 and KA2 subunits exhibit high ligand affinity.^46–50 Using in situ hybridization studies, KA receptor subunits have been detected in mouse, rat, and cat eyes.^13,37 Interestingly, KA1 subunit was found in mouse but not in rat retina.^13,35 Various KA receptor subunits were found in rat retina in the inner nuclear layer and ganglion cell layer.^51,52 Using GluR6 and GluR7 antiserum, immunocytochemistry studies confirmed localization of KA receptors in the mammalian retina (horizontal, amacrine, ganglion, and possibly bipolar cells).^51,52

In the retina, glutamate that is released from cones activates KA receptors on bipolar cell dendrites, leading to influx of Na⁺, K⁺ and consequent depolarization of the neuronal membranes.⁵³ Darkness-mediated neurotransmitter release from the cones has been reported to desensitize KA receptors.⁵⁴ A number of studies support the hypothesis that KA receptors exhibit a dual signaling system, consisting of a slow graded response that modulates the rapid, AMPA receptor–mediated actions^55–57 and rapidly rising and decaying responses at other sites.^58,59 Activation of kainite receptors in retina elicits pathological excitotoxic changes, in vivo and in vitro, suggesting a potential neuroprotective role for these receptors in these tissues.⁶⁰

Metabotropic Glutamate Receptors

mGluR subunits form a functional receptor as a single protein coupled to membrane-bound G-proteins. Eight mGluRs grouped into 3 groups have been cloned; Group I mGluRs (mGluR1, mGluR5) activate Phospholipase C; Group II mGluRs (mGluR2, mGluR3) inhibit adenylyl cyclase; and Group III mGluRs (mGluR4, mGluR6, mGluR7, mGluR8) inhibit adenylyl cyclase.^61–67 Table 1 provides an overview of the localization of mGluRs in the retina. The mGluRs are also involved in synaptic transmission between photoreceptors and ON bipolar cells which express group III mGluR.^86,87 Immunocytochemistry studies detected mGluR6 in ON bipolar cells in outer plexiform layer of rat retina but not the mGluR3 subtype.^75,79

Table 1.

Overview of the Localization of Metabotropic Glutamate Receptors in the Retina

Retinal cell	Receptors
	Group I		Group II			Group III
	mGluR1	mGluR5	mGluR2	mGluR3	mGluR4	mGluR6	mGluR7	mGluR8
Photoreceptors	+^70,78,82	+^78,82	n/a	n/a	n/a	n/a	n/a	+^66,76,77
Horizontal cells	+^73,82	+⁷³	+^70,73	n/a	n/a	n/a	+⁷³	+⁷⁷
Bipolar cells	+^73,74,82	+^72,74,82	n/a	+⁷¹	n/a	+^{64,68,72,79,84,85}	+^68,69,73	+⁶⁶
Amacrine cells	+^{70,72,74,78,82}	+^74,78,82	+^70,72,75,80	n/a	+^72,75,81	n/a	+^68,69,72,81	+^66,76,77,83
Ganglion cells	+^70,72,78	+^78,83	+^72,80,83	+⁸³	+^68,72,75,83	+⁸³	+^68,72,83	+^66,76,77,83

+, localized; n/a, data not available.

Group I mGluRs, which are coupled to G_q, mobilize intracellular calcium and activate protein kinase C (PKC) through activation of Phospholipase C and mediate hydrolysis of phosphoinositides to form inositol trisphosphate (IP₃) and diacylglycerol.⁸⁸ Activation of a calmodulin-positive mGluR1 in white bass amacrine cells reduced sensitivity of GABA_A receptor due to release of intracellular Ca²⁺ ions.⁸⁹ In vertebrate amacrine cells, the enhanced current from GABA was dependent on both intracellular Ca²⁺and PKC following activation of mGluR5.⁷⁸ While Group II and Group III mGluRs are coupled to G_i and can activate K⁺ channels, these receptors may inactivate Ca²⁺ channel through inhibition of adenylyl cyclase.⁸⁸ Group II mGluRs have been reported to influence the directional selectivity in RGCs presumably due to its ability to inhibit acetylcholine and GABA release from amacrine cells.⁹⁰ Group III mGluR reduces inwardly rectifying K⁺ channel current due to phosphorylation by cGMP-dependent kinase.⁹¹ While Group 1 GluRs can potentially enhance excitotoxicity due to increase in release of intracellular Ca²⁺ ions, Groups II and III mGluRs could exert a neuroprotective action due to their ability to inhibit the cyclic AMP pathway.^92–95

Ischemia, Hypoxia, Hypoglycemia, Excitotoxicity

Experimental approaches to the study of neuroprotection in the retina have used insults that trigger ischemia, hypoxia, hypoglycemia, or excitotoxicity. Ischemia is characterized by insufficient blood supply that results in compromised ability to meet cellular energy requirements.⁹⁶ While hypoxia and hypoglycemia are components of ischemia, they are distinct from ischemia.³³ However, each of these noxious stimuli can trigger excitotoxic cascade in neurons.^33,97–100 Excitotoxicity refers to overstimulation of the NMDA receptors consequent to excessive accumulation of glutamate in the extracellular spaces in the neurons, ultimately causing cell death.^30–33 Moreover, excitotoxicity has been postulated to present a common downstream mechanism for several stimuli, including hypoglycemia, hypoxia, ischemia, trauma, and chronic neurodegenerative diseases, that terminate in cell death.³² To this end, excitotoxicity has been implicated in several conditions associated with chemical and pathological changes in neuronal injury, including stroke, spinal cord trauma, and head injury as well as in degenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, among others.^101,102

Several mechanisms have been implicated in neuronal damage caused by excitotoxicity. Excessive glutamate concentrations trigger a neurotoxic cascade involving several mechanisms, including elevation of intracellular Ca²⁺ and the activation of AMPA/kainite and NMDA receptors. Glutamate hyperactivity also induces overload of Ca²⁺ and Na⁺ ions in postsynaptic neurons.¹⁰³ The increase in intracellular Ca²⁺ results from reduced mitochondrial sequestration and reduced binding to intracellular Ca²⁺ binding proteins or influx through voltage-gated Ca²⁺ channels.^104–107 This elevated intracellular Ca²⁺ can induce Ca²⁺ release from endoplasmic reticulum, alter mitochondrial membrane permeability, and activate both caspase-dependent apoptosis and mitochondrial dehydrogenases leading to the formation of free radicals^108,109 and the nitric oxide–induced neurotoxic cascade,³² ultimately, culminating in neuronal death. Since excitotoxicity represents a common downstream pathway for ischemia, hypoxia, and hypoglycemia, this review will further discuss the role of excitotoxicity in retinal neurodegeneration, a well-accepted concept in the etiology of glaucomatous optic neuropathy (GON).

Excitatory amino acid–mediated excitotoxicity in retina, in vivo

Animal models have been used to investigate excitatory amino acid–induced excitotoxicity and the neuroprotective actions and mechanisms of potential neuroprotective drugs. Due to the abundant expression of glutamate receptors, many studies have focused on the role of these receptors in excitotoxicity. In an in vivo rat model, a single intravitreal injection of the glutamate receptor agonist, NMDA, induced visual behavioral changes that were abolished by coadministration of the nonselective NMDA-receptor-channel antagonist, MK801.¹¹⁰ Similarly, chronic intravitreal injection of low doses of glutamate elicited RGC degeneration that was partially mitigated by the NMDA receptor antagonist, memantine,¹¹¹ implying a significant role for glutamate receptors in retina toxicity. To elucidate the role of glutamate receptors in RGC apoptosis, Guo et al. assessed the neuroprotective potential of selective NMDA antagonist (ifenprodil) and nonselective NMDA antagonist (MK801), as well as Group II mGluR agonist (LY354740) in staurosporine (SS)-induced RGC death in Dark Agouti rats, in vivo.¹¹² Intravitreal injection of each of these glutamate receptor antagonists attenuated RGC apoptosis with the following EC₅₀ values: MK801 (EC₅₀ = 0.074 nM), ifenprodil (EC₅₀ = 0.0138 nM), and LY354740 (EC₅₀ = 19 nM), affirming the role of glutamate receptors in SS-mediated RGC apoptosis, in vivo. Similarly, intravitreal injection of the most potent combination of receptor antagonists (as assessed in SS-mediated insult rat model), MK801 (0.06 nM) and LY354740 (20 nM), protected RGC apoptosis in a chronic ocular hypertension rat model, in vivo.¹¹² Taken together, these data point to the involvement of both NMDA and non-NMDA receptors in RGC degeneration, in vivo.

In addition to the glutamate receptor antagonists, other compounds have been evaluated for neuroprotection in animal models. Rácz et al. reported a neuroprotective action for pituitary adenylate cyclase activating polypeptide (PACAP) against monosodium glutamate–mediated insult in rat retina, in vivo.¹¹³ PACAP attenuated pro-apoptotic signaling factors, caspase-3, c-Jun N-terminal kinase (JNK), apoptosis inducing factor, and cytochrome c in newborn Wistar rat pups, while anti-apoptotic signaling factor, phospho-Bcl-2-associated death promoter (Bad), was elevated, in vivo. Moreover, these effects were reversed by the PACAP antagonist, PACAP6-38, thereby affirming the degenerative action of monosodium glutamate in activating apoptotic pathways rat retina, in vivo, and the protective role of PACAP in retina.¹¹³

The neuroprotective role of PACAP in the retina was further corroborated by D'Amico et al. in streptozotocin-induced diabetic retinopathy (DR) rat model. These investigators reported a downregulatory action for PACAP on the expression of pro-angiogenic factors, hypoxia-inducible factors (HIF)-1α and −2α, while that of the anti-angiogenic factor, HIF-3α, was upregulated, in vivo.¹¹⁴ Moreover, intravitreal injection of PACAP similarly downregulated the expression of VEGF and its receptors in streptozotocin-induced DR rat model, suggesting a protective function for PACAP in retina, in vivo.¹¹⁵

Sakai et al. examined the effect of intravitreal injection of NMDA (25 mM) on retina in glutathione peroxidase 4 (GPx4)^+/+ and GPx4^+/− mice. Interestingly, mice with defective expression of GPx4 exhibited higher lipid peroxidation (assessed by 4-HNE immunostaining), higher apoptosis (as measured by TUNEL staining), and low ganglion cell layer cell density. In addition to corroborating the apoptotic role of NMDA in retina degeneration, in vivo, these studies point to the potential role of antioxidants in mitigating NMDA-induced excitotoxicity.¹¹⁶

In other studies, intravitreal injection of glutamate (500 nM) induced retinal apoptosis in Wistar rats, in vivo. Interestingly, these deleterious effects were attenuated by intravitreal pretreatment of animals with neuropeptide Y (NPY, 2.35 nM).¹¹⁷ More recently, ischemia/reperfusion injury was reported to elicit degradation of the thickness of retinal layers (whole retina layer, inner plexiform layer, and inner nuclear layer) and a reduction in number of cells within the ganglion cell layer in a mouse model.¹¹⁸ Furthermore, pretreatment with the JNK inhibitor, SP600125, for 2 h protected mouse retina from these deleterious actions of ischemia/reperfusion.¹¹⁸

In summary, the ability of an excessive glutamate receptor mediated signal to induce excitotoxic damage to retinal neurons, in vivo, is well established. More importantly, the observation that peptides such as PACAP and NPY can ameliorate the glutamate-induced neurotoxicity is intriguing and should be a subject of further investigation. Table 2 summarizes the neuroprotection in the retina under in vivo conditions.

Table 2.

Neuroprotection in Retina Under In Vivo Conditions

Animal model	Stimuli	Pharmacological action	Neuroprotection strategy
Adult rats	Intravitreal NMDA	RGC degeneration; impaired vision	Coadministration of the nonselective NMDA antagonist, MK801, abolished visual alterations¹¹⁰
Adult rats	Intravitreal glutamate	RGC degeneration	Intraperitoneal injection of memantine partially protected RGCs¹¹¹
Dark Agouti rats	Intravitreal staurosporine	RGC apoptosis	Coadministration of glutamate receptor antagonists attenuated RGC apoptosis as follows: MK801 (EC₅₀ = 0.074 nM), ifenprodil (EC₅₀ = 0.0138 nM), and LY354740 (EC₅₀ = 19 nM)¹¹²
Dark Agouti rats	Ocular hypertension	RGC apoptosis	Intravitreal injection of combined regimen: low-dose MK801 (0.06 nmoles) and LY354740 (20 nmoles) maximally attenuated RGC apoptosis (72 ± 9 compared with MK801 alone at 64 ± 12)¹¹²
Newborn Wistar rats	Subcutaneous monosodium glutamate	Retinal degeneration characterized by: stimulation of caspase-3, JNK, apoptosis inducing factor, and cytochrome c, but reduction of Bad phosphorylation	Intravitreal injection of PACAP attenuated pro-apoptotic signaling factors, caspase-3, JNK, apoptosis inducing factor, and cytochrome c; anti-apoptotic signaling factor, phospho-Bad was elevated¹¹³
3-month-old male Sprague–Dawley rats	Intraperitoneal streptozotocin	Retinal degeneration in early stage of hyperglycemia	Intravitreal injection of PACAP modulated HIF (downregulated HIF-1α and −2α expression and upregulated HIF-3α expression)¹¹⁴
Adult male Wistar rats	Intravitreal glutamate	Retinal degeneration, especially in the inner nuclear layer and ganglion cell layer	Intravitreal pretreatment with NPY 2 h before glutamate injection caused 55% reduction in TUNEL-positive cells and increase in Brn3a-positive cells in retinal slices¹¹⁷
GPx4^+/+ and GPx4^+/− mice	Intravitreal NMDA	Greater lipid peroxidation and apoptosis in (GPx4^+/−) mice, compared to GPx4^+/+ mice	Presence of GPx4^+/+ conferred protection from NMDA¹¹⁶
C57BL/6J mice	Ocular hypertension	Retinal degeneration characterized by reduction in thickness of whole retina, inner plexiform, inner nuclear, and ganglion cell layers	Intravitreal pretreatment with JNK inhibitor, S600125, 2 h before IOP elevation attenuated retinal degeneration¹¹⁸

Bad, Bcl-2-associated death promoter; EC₅₀, effective concentration that induces 50% response; GPX4, glutathione peroxidase 4; HIF, hypoxia-inducible factors; JNK, c-jun N-terminal kinase; NMDA, N-methyl-d-aspartate; NPY, neuropeptide Y; PACAP, pituitary adenylate cyclase activating polypeptide; RGC, retinal ganglion cell; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling.

Excitotoxicity in the retina, in vitro: Roles of glutamate, hypoxia, and hypoglycemia

Using [³H]-d-aspartate as a marker for glutamatergic neurotransmission, exogenously administered glutamate has been shown to evoke the release of glutamate from chick retina.^98,119 Hypoxia and/or hypoglycemic stimuli have also been reported to induce release of glutamate in retinal neurons.^31,120 There is evidence that hypoglycemia and hypoxia can induce excitotoxicity by distinct mechanisms in some tissues. For instance, hypoxia induced selective retina ganglion cell death in pig retinal cultures, while hypoglycemic stimuli led to death and reduction in the number of photoreceptors, bipolar, amacrine, and RGCs.¹²¹ Thus, glutamate-, hypoglycemia-, and hypoxic-stimuli represent viable models for studies of neurotransmitter release that can be used to investigate the potential neuroprotective actions of drugs in retina, in vitro.

Glutamate-induced [³H]-d-aspartate release

In a follow-up to experiments performed on the chick retina by Lopez-Colome et al.,¹¹⁹ Ohia et al. characterized the effect of l-glutamate on the release of radiolabeled excitatory neurotransmitter (represented by [³H]-d-aspartate) in mammalian retina using superfusion experiments.¹²² The broad spectrum GluR agonist, l-glutamate, but not its enantiomer, d-glutamate, evoked [³H]-d-aspartate release from isolated bovine retina. The effect of l-glutamate on the neurotransmitter release was species dependent, with equimolar concentrations of l-glutamate exhibiting comparable effects in bovine and human retina but with a relatively weak action in the rabbit retina.¹²²

Since the release of amino acids from retinal neurons can occur by both Ca²⁺-dependent and Ca²⁺-independent mechanisms,^32,98 Ohia et al. examined the role of Ca²⁺ on l-glutamate–induced [³H]-d-aspartate release in the mammalian retina. Inhibition of N- and L-calcium channels (nitrendipine; omega-conotoxin) and omission of calcium from buffer partially blocked l-glutamate–induced [³H]-d-aspartate release in isolated bovine retina, suggesting that both Ca^2+-dependent and Ca²⁺-independent pathways account for the release of [³H]-d-aspartate in bovine retina.¹²² Consistent with reports from Ohia et al., the L-type Ca²⁺ channel, diltiazem was found to attenuate glutamate-induced excitotoxicity in a newborn rat retinal cell culture model.¹²³

Toriu et al. also reported that exclusion of Ca²⁺ from the medium eliminated glutamate-induced toxicity in rat cultured retinal neurons.¹²⁴ In the same studies, the T- and L-Ca²⁺ channel blocker, lomerizine, protected cultured retinal neuron from glutamate-induced toxicity in a concentration-dependent manner.¹²⁴ In contrast, Calzada et al. examined the effects of different calcium channel blockers in an in vitro model of NMDA-evoked RGC excitotoxicity in rabbit explants. The Ca²⁺ channel blockers, verapamil (L- and T-channel blocker), nimodipine (L-channel blocker), or omega-conotoxin (N, P, and Q channel blockers) had no effect on NMDA-induced toxicity in the retina explants.¹²⁵ These studies support the notion that excitotoxicity may occur by both Ca^2+-independent and Ca²⁺-dependent mechanisms.^32,98 It is also pertinent to note that factors, such as the animal species, the nature of excitotoxic stimuli, the types of Ca²⁺-channels, and in vivo versus in vitro models, could account for the different results reported. Taken together, these data reflect the complexity of the role of Ca²⁺ in experimental excitotoxicity.

Ohia et al. further delineated the role of several GluR agonists on basal [³H]-d-aspartate release in the retina. Both metabotropic and ionotropic receptor agonists evoked [³H]-d-aspartate release in the retina. Compared to l-glutamate and l-aspartate, NMDA displayed the least potency, presumably due to its selectivity for the ionotropic NMDA-glutamate receptor subtype, in contrast to glutamate which activates all glutamate receptor subtypes. Of the mGluR agonists investigated, the Group III and Group II agonists (L-Ap4 and DCG-IV) were more potent in bovine retina, while Group I and Group I/II (quisqualate and trans-ACPD) were more potent in human retina, suggesting a species-dependent distribution of glutamate receptor subtypes.¹²² Interestingly, Group I mGluR has been reported to potentiate neuronal excitability,^126,127 while Groups II and III mGluRs attenuate glutamate release in the CNS.^128,129 Thus, GluRs can play a significant role in regulating glutamate availability in the nervous system.

Since the NMDA receptor possesses a recognition site for polyamines,¹³⁰ the effect of polyamines, spermine, and spermidine on l-glutamate–induced [³H]-d-aspartate release was evaluated. Both spermine and spermidine enhanced the neurotransmitter release, corroborating their role as positive modulators of the NMDA receptor.^130–132 Antagonists at the NMDA-receptor (MK-801; AP-5), metabotropic receptor (MCPG; Group I/II), and polyamine site (ifenprodil; arcaine) reversed l-glutamate–induced [³H]-d-aspartate release in a manner similar to that reported for these antagonists in brain tissue.^133,134 Interestingly, while ifenprodil partially protected, MK-801 completely protected chick retina against NMDA-induced toxicity.¹³⁵ In support of these observations, Vallazza-Deschamps et al., demonstrated a protective action for MK-801 and CNQX (AMPA/KA receptor antagonists) from glutamate-induced toxicity in porcine and newborn rat retinal cultures.¹²³ Thus, these receptor sites constitute pharmacological targets for potential neuroprotective actions in the retina.

In addition to the l-glutamate (represented by [³H]-d-aspartate) induced excitotoxicity demonstrated in the superfusion studies described above, other investigators have also utilized the amino acid–induced excitotoxicity model to evaluate the neuroprotective actions of nicotinic acetylcholine receptor (nAChR) agonists and NPY. Using immunocytochemical techniques, Thompson et al. described α4 and β2 nicotinic acetylcholine receptor subtypes in small porcine RGCs and the α7 receptor subtype in large porcine RGCs. These investigators further demonstrated a neuroprotective action for α_4-nAChRs in isolated pig RGCs from glutamate-induced excitotoxicity with the following rank order of activity: epibatidine > anatoxin > UB165 > RJR2403 > lobeline HCl.¹³⁶ In corroboration, the α₄-nAChR antagonist, dihydro-beta-erythroidine, reversed the protective effects of α₄-nAChRs agonists.¹³⁶

In addition to the α4-nAChR subtype, there is evidence supporting a neuroprotective role for the α7-nAChR subtype in glutamate-induced toxicity in mammalian retina.^137,138 In adult rat retina, nicotine, acetylcholine, or the potent, selective α7 nAChR agonist, PNU-282987 protected RGCs from glutamate-induced excitotoxicity.¹³⁸ Similarly, α7-nAChR agonist, tropisetron, protected adult porcine RGCs from glutamate-induced excitotoxicity, presumably through mechanisms that involved a reduction in p38 MAPK signaling pathway and an internalization of the NMDA receptors.¹³⁷ It is of interest to note the role of nicotinic receptors in preventing glutamate-induced neurotoxicity in these in vitro models. However, the mechanism/s involved in the nicotinic receptor-mediated pathway associated with this response is yet to be fully determined.

The neuroprotective function of NPY from glutamate-induced excitotoxicity in brain slices has been described by several investigators.^139–143 Thus, Santos-Carvalho et al., sought to determine the role of NPY in retinal degeneration. Activation NPY₂,_4,5 mitigated glutamate-induced necrotic cell death, while only NPY₅ receptors attenuated apoptotic cell death in rat retinal cells.¹¹⁷ Moreover, the neuroprotection was achieved by mechanisms involving the PKA and p38K signaling pathways.¹¹⁷ In a recent study, Martins et al., reported that activation of NPY₁ receptors mitigated NMDA-induced elevation in intracellular Ca²⁺ in purified rat RGCs. Similarly, NPY inhibited NMDA-induced degeneration in rat retinal explants, in vitro.¹⁴⁴

In summary, the use of in vitro studies to investigative the neuroprotective pathways in the retina reveals the role for glutamate, nicotinic, and NPY receptors in modulating excitotoxicity induced by the excitatory amino acid, glutamate. Taken together, it appears that there are multiple mechanism/s for prevention of damage caused by excessive glutamate concentrations in the retina. It is feasible that several pathways are involved in protecting the retina from toxic insults in ocular neurodegenerative conditions such as glaucoma, DR, and retinal ischemia.

Hypoxia induced [³H]-d-aspartate release

In 1994, Neal et al. showed evidence that hypoxia can enhance aspartate and glutamate release from both rabbit and rat retina.³¹ Similar to Neal et al., Ohia et al. reported that hypoxia induced [³H]-d-aspartate release from isolated bovine retina by mechanisms that were partially reversed by the Ca^2+-channel blockers (diltiazem, nitrendipine, verapamil, and ω-conotoxin), suggesting the partial involvement of Ca²⁺ in the release process.¹⁴⁵ Moreover, the magnitude of hypoxia-induced release was comparable in both bovine and human isolated retina.

The endogenous, broad spectrum agonist, l-glutamate, enhanced while KA attenuated hypoxia induced [³H]-d-aspartate release, suggesting a unique role for the KA iGluRs in hypoxia-induced neurotransmission in isolated bovine retina. Similar to the observation made by Ohia et al.¹²² on the effect of NMDA on basal [³H]-d-aspartate release, NMDA (up to 1 mM concentration) had no effect on hypoxia-induced neurotransmitter release, suggesting that hypoxia and glutamate release processes may occur by similar mechanisms. Taken together, these observations imply that NMDA receptors are not involved in hypoxia-mediated neurotransmitter release in this study. Since the detrimental effects of hypoxia are time dependent,³³ it is conceivable that the exposure time applied for NMDA was not sufficient to activate the iGluRs.

To delineate the role of GluRs involved in the hypoxia-mediated release from bovine retina, the effects of various GluR antagonists and NMDA receptor modulators were investigated by Ohia et al.¹⁴⁵ These workers found that NMDA-receptor (MK-801), metabotropic receptor [L-AP3 (Group I); MCPG (Group I/II)], and polyamine site (ifenprodil) antagonists inhibited hypoxia–induced [³H]-d-aspartate release with the following rank order of potency: ifenprodil ≈ MCPG > L-Ap3 > MK-801.¹⁴⁵ It was not surprising that the NMDA receptor antagonist MK-801 exhibited the least effect, supporting the finding that, under these experimental conditions, iGluRs minimally contribute to hypoxia–induced [³H]-d-aspartate release in bovine retina.

In contrast to the observation demonstrated in the glutamate-induced [³H]-d-aspartate release model in which the IC₃₀s for antagonists were observed in the micromolar range,¹²² the hypoxic model was highly sensitive to inhibition by these antagonists (IC₃₅ in nanomolar range).¹⁴⁵ Indeed, the protective role of glutamate receptors on hypoxia-induced neurotransmitter release has also been reported for hippocampal rat slices^146,147 and cultured RGCs.¹⁴⁸ Interestingly, L-AP3 (mGluR antagonist) but not ifenprodil, MCPG, and MK-801 attenuated hypoxia-induced neurotransmitter release in human retina,¹⁴⁵ suggesting a prominent role for mGluRs in hypoxia excitotoxicity in human retina.

Hypoglycemia–induced [³H]-d-aspartate release

There is evidence that hypoglycemia can elicit degeneration of primary retina cells.^121,149 Ohia et al., investigated the pharmacological actions of potential neuroprotective agents on hypoglycemia–induced [³H]-d-aspartate release in isolated bovine and human retina.¹⁵⁰ These researchers found that hypoglycemia–induced [³H]-d-aspartate release was partially sensitive to inhibition by L-type (diltiazem, nitrendipine) and N-type (ω-conotoxin) channel blockers and Ca²⁺-free buffer in a manner similar to those observed with the glutamate–induced¹²² and hypoxia–induced¹⁴⁵ [³H]-d-aspartate release models. With exception of KA-iGluR, the broad spectrum agonist, glutamate, and NMDA iGluR agonist, NMDA, increased hypoglycemia–induced [³H]-d-aspartate release, suggesting that the NMDA- and KA-receptor iGluRs play different roles in hypoxia- and glutamate-induced release.¹⁵⁰

To determine the potential neuroprotective role of GluRs in the hypoglycemia-mediated release from bovine retina, the effects of various GluR antagonists and NMDA-receptor modulators: noncompetitive, MK-801 (nonselective NMDA-receptor); MCPG (metabotropic receptor Group I/II); L-AP3 (Group I); ifenprodil, arcaine (polyamine site) were investigated.¹⁵⁰ It was interesting to note that MCPG, ifenprodil, and L-AP3 attenuated hypoglycemia-induced neurotransmitter release with IC₅₀s in the micromolar range.¹⁵⁰ Moreover, at an equimolar concentration (10 μM), the L-AP3 and MK-801 were most potent (P < 0.0001 and 0.001, respectively), while arcaine and ifenprodil exhibited no effect.¹⁵⁰ Taken together, these observations suggest a potential neuroprotective function for these GluRs in hypoglycemia-induced excitotoxicity. Table 3 summarizes the pharmacological activity of glutamate receptor agonists/antagonists on the release of excitatory amino acids from mammalian retina under in vitro conditions.

Table 3.

Comparative Inhibitory Concentration Values for Glutamate Receptor Inhibitors/Modulators in Suppressing [³H]-d-Aspartate Release from Retina In Vitro and Other Functional Responses

Antagonists	IC₃₀ (μM)Inhibition of d-glutamate–induced release ¹²²	IC₃₅ (nM)Inhibition of hypoxia–induced d-aspartate release ¹⁴⁵	IC₅₀ (nM)Inhibition of hypoglycemia-induced release ¹⁵⁰	IC₅₀ (nM)Inhibition of functional responses in the neurons
MCPG	0.35 ± 0.09	2.98 ± 0.59	1.98 ± 0.76	370 ± 70¹⁵³
Ifenprodil	0.72 ± 0.16	2.80 ± 0.64	19.05 ± 1.30	0.0138¹¹²
AP-5	1.23 ± 0.66	17.19 ± 2.29	24.08 ± 1.42	8.29 ± 0.63¹⁵⁴
Arcaine	4.90 ± 0.99	n/a	n/a	55¹⁵⁵
MK-801	11. ± 1.94	n/a	n/a	0.074¹¹²

IC, inhibitory concentration.

Summary and Conclusion

As the “baby boomers” advance in age, ocular neuropathies are projected to increase significantly in the next few years.¹⁵¹ So far, the translation of neuroprotection strategies into clinical applications that can successfully mitigate the impact of ocular neuropathies have remained elusive. Glutamate receptors are abundantly expressed in the mammalian retina, conferring a high susceptibility to excitotoxicity by this amino acid when present at high concentration in the synaptic and extrasynaptic locations. It is pertinent to note that several receptors other than those for glutamate (such as those for nicotine and some neuropeptides) play an important role in preventing retinal neuron from toxic insults.

Since excitotoxicity presents a common pathway in multiple degenerative conditions, including ocular neuropathies such as glaucoma and DR, the employment of therapeutic strategies that target these pharmacological receptors is needed to unravel their impact on processes involved in neuronal viability. Thus, both in vivo and in vitro experimental models of excitotoxicity described in this review article remain a viable option that can be utilized in the initial screening of potential neuroprotective drugs. Techniques involving use of NMR imaging to monitor endogenous levels of retinal ATP before, during, and after hypoxic/ischemic insults that mimic what happens in the retina during poor ocular blood flow may also be useful tools.¹⁵² The ability of various drugs, such a MK-801 and diltiazem, to preserve retinal ATP levels or to help recover this important mitochondrial-derived energy source could then be screened.¹⁵² Further progress in finding therapeutic agents to ameliorate RGC cell death as in GON is eagerly awaited.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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Regulation of Excitatory Amino Acid Transmission in the Retina: Studies on Neuroprotection

Abstract

Abstract

Introduction

Glutamate receptors in retina

Ionotropic Glutamate Receptors

NMDA receptors

AMPA receptors

KA receptors

Metabotropic Glutamate Receptors

Ischemia, Hypoxia, Hypoglycemia, Excitotoxicity

Excitatory amino acid–mediated excitotoxicity in retina, in vivo

Excitotoxicity in the retina, in vitro: Roles of glutamate, hypoxia, and hypoglycemia

Glutamate-induced [3H]-d-aspartate release

Hypoxia induced [3H]-d-aspartate release

Hypoglycemia–induced [3H]-d-aspartate release

Summary and Conclusion

Footnotes

Author Disclosure Statement

References

Glutamate-induced [³H]-d-aspartate release

Hypoxia induced [³H]-d-aspartate release

Hypoglycemia–induced [³H]-d-aspartate release