Glutamate Receptor Signaling in Retina Müller Cells: Plausible Role in Neurodegeneration

Abstract

The retinal network relies on glutamate, the primary excitatory neurotransmitter involved in the visual cycle. Glutamate transactions are carried out by an array of distinct receptors and transporters distributed across both pre- and post-synaptic neurons and Müller radial glial cells. Glutamate receptors are broadly divided into two types: ionotropic and metabotropic receptors that differ in their molecular architecture and signaling properties. Within the retina, Müller glia cells span across its entire layers and possess specialized features that enable them to regulate glutamate extracellular levels and thus, its neuronal availability. In order to prevent an excitotoxic insult, retina extracellular glutamate levels have to be tightly regulated through uptake, predominantly into Müller glial cells, by a family of Na⁺-dependent glutamate transporters known as excitatory amino acid transporters. An exquisite interplay between glutamate receptor signaling and glutamate transporter expression and function is fundamental for the integrity and proper function of the retina. This review examines our current understanding of the impact of Müller glial glutamate signaling on glia/neuronal coupling.

1. Introduction

The retina is a delicate and vital part of the central nervous system (CNS) which contributes to our understanding of the brain with its surprisingly intricate cellular architecture [1]. This structure is a complex mosaic encompassing five distinct classes of specialized neurons dedicated to processing information received by the eye [2,3,4]. Retinal neurons—namely photoreceptors, bipolar cells, ganglion cells, horizontal cells, and amacrine cells—exhibit a characteristic distribution across the retinal layers [1,2]. Retinal neurons are interconnected through the vertical and horizontal pathways collaboratively contributing to the processing of visual images and the subsequent transmission of this information to the brain via the optic nerve [3]. The horizontal pathway involves lateral connections between horizontal cells and amacrine cells, facilitating feedback signals between photoreceptors and bipolar cells (for horizontal cells) and between bipolar cells and retinal ganglion cells (RGCs) (for amacrine cells) [5]. Conversely, the vertical pathway initiates the transduction of light signals by photoreceptors and bipolar cells, culminating in the transmission to the brain through the axons of RGCs (Figure 1). The excitatory neurotransmitter glutamate (Glu) is the most important signal in this pathway. Across retinal neurons, various neurotransmitter receptors are diversely expressed and this contributes significantly to functional diversity, forming the foundation for multiple processing pathways of visual information. Two primary classes of receptors mediate the effects of Glu: ionotropic receptors (GRI) and metabotropic receptors (GRM) [6]. A plethora of factors regulate the activities of these receptors such as subunit composition, density, receptor affinity, and localization at the synapse [7].

The retina also comprises non-neuronal cells such as astrocytes, microglia, and Müller cells (MCs) that support retinal neurons. Astrocytes are confined to the innermost layers of vascularized retinas. In contrast, MCs are radially shaped glial cells that extend across the entire thickness of the retinal neurons establishing close connections and contributing significantly to the preservation of tissue structure, homeostasis, and light transmission (Figure 1) [8,9]. MCs play a crucial role in sustaining the functionality and metabolism of retinal neurons, actively contributing to both normal retinal function and the processes associated with retinal degeneration [10]. MCs constitute a now accepted, indispensable component of a robust and optimally functioning retina [11]. Disruptions in these cells inevitably perturb cellular communication within the retina, compromising its proper function. The precision of metabolic processes in MCs can be quantified and is an actively explored area. Investigating the factors triggering metabolic changes in disease and understanding the impact of these changes on MCs’ ability to support neuronal function is crucial for comprehending the progression of retinal disease and fundamental for designing therapeutics compatible with the diseased retina. Despite their significance, MCs remain a relatively understudied cell type despite their role in excitotoxicity and neurodegeneration. In this contribution, we seek to provide a comprehensive overview of Glu receptor expression and signaling in MCs and delineate the MC’s role in the prevention of excitotoxicity events occurring in the retina.

2. Glu-Mediated Neurotransmission in the Retina

In the retina, the process whereby light energy is converted into visual information is known as phototransduction; this activity is modulated by a diverse array of retinal cells, neurotransmitters, transporters, and receptors [12]. The phototransduction process is made up of four sequential steps. Photoreceptors are the first point of contact with light rays and they are made of two types: rods and cones. Photoreceptor cells possess different features such as chromatic sensitivity and relative abundance. Moreover, the photoreceptors’ relative abundance and chromatic sensitivity, rods and cones play a critical role in light sensitivity. An increase in light reduces the sensitivity of rods thereby activating the cones for the photopic vision. Yin et al. [13] showed that a mixture of signals between the rods and cones occurs as illumination increases from nearly 100% of rods in the low-mesopic range to about 20% in the mid-level photopic range. The activation of rod signals in the low-mesopic range to the relatively high photopic range in the cone visual pathway experiment conducted in retinal ganglion cells was shown to be an adaptive mechanism for varying illumination signals hence emphasizing the critical role of photoreceptors in the visual pathway [14].

In the light cycle, light absorption occurs when photons hit the photoreceptors and it is captured by the visual photopigments (rhodopsin) present in rods and cones [15,16]. In response to light conditions, photons are absorbed by rhodopsin and cis-retinal converted into an isomerized form (all-trans-retinal). The conformational change allows the disintegration of the opsin from the retinal, adopting an unstable conformation called “metarhodopsin”. cGMP is hydrolyzed by the transducin α subunit, a G-protein, activated by metarhodopsin. Once activated, it stimulates a cGMP phosphodiesterase (PDE) [17,18] leading to the closure of Na⁺ channels thereby hyperpolarizing the cells [16]. This leads to a shift in the membrane potential from −40 mV to −75 mV [16]. Due to this, K⁺ channels open and Ca²⁺ channels close [19], causing the vesicles loaded with glutamate (Glu) to be trapped within the presynaptic axon terminals [20], reducing the amount of Glu released [21]. This amino acid is widely considered the primary excitatory neurotransmitter in the retina [22] and plays a crucial role in the forward transmission of visual signals among photoreceptors, bipolar and ganglion cells [23,24].

Under dark conditions, photoreceptors are depolarized due to the opening of cGMP-dependent Na⁺ channels. The increase in intracellular Ca²⁺ concentrations causes the synaptic vesicles loaded with Glu to move to the active zone and fuse with the membrane, then Glu is released into the synaptic cleft [20,25,26].

Consequently, bipolar cells serve as the site for the separation of ON and OFF signaling [16]. OFF bipolar cells, equipped with ionotropic Glu receptors, depolarize in the dark in response to the Glu released by photoreceptors [27,28]. On the other hand, ON bipolar cells express metabotropic Glu receptors, specifically mGluR6, on their dendrites [29]. The activation of mGluR6, linked to the G protein Go, triggers a downstream second-messenger system, resulting in hyperpolarization of the cell following Glu binding [30,31]. Therefore, ON bipolar cells exhibit hyperpolarization in the dark, contrasting with the photoreceptors and OFF bipolar cells [32]. Photoreceptors and bipolar cells are unique in their lack of action potential generation and response to light through graded potentials, thereby influencing the release of Glu [33]. In the visual system, synaptic ribbons are also present in the output synapses of second-order retinal bipolar neurons, which, like retinal photoreceptors, auditory hair cells, and pineal photoreceptors, signal through graded changes in membrane potential [34].

Physiological investigations with isolated cells reveal that only micromolar levels of Glu are necessary to activate Glu receptors [35,36]. Consequently, the quantity of Glu released into the synaptic cleft exceeds the concentration needed to activate most postsynaptic receptors by several orders of magnitude. To prevent cell death, high-affinity Glu transporters known as excitatory amino acid transporters (EAATs) located on adjacent neurons and surrounding MCs swiftly eliminate Glu from the synaptic cleft [37]. Five cloned Glu transporters, namely EAAT-1 (GLAST), EAAT-2 (Glt-1), EAAT-3 (EAAC-1), EAAT-4, and EAAT-5, play pivotal roles in this process [38,39,40]. Neuronal Glu transporters include the excitatory amino acid carrier (EAAC-1), EAAT-4 and EAAT-5 [41]. EAAC-1 is predominantly found in amacrine, ganglion, and horizontal cells. EAAT-4 has been identified in retinal astrocytes and the retinal pigment epithelium [42,43]. EAAT-5 is well expressed in photoreceptors, bipolar, and amacrine cells and is characterized by low Glu transport activity and significant anion conductance, functioning primarily as a Glu-activated chloride channel to modulate neuronal excitability [44]. Notably, a splice variant of Glt-1/EAAT-2, known as Glt-1c, is selectively expressed in photoreceptor axon terminals [45].

Distinct from both iGluRs and mGluRs, Glu transporters exhibit unique pharmacological characteristics. Substrates for the transporters include L-Glu, L-aspartate (Asp), D-Asp [46,47], and L-Homocysteate (L-HCA) [48]. While Glu receptor agonists and antagonists are not recognized [49,50]. Upon uptake, Glu transporters incorporate Glu into MCs, concomitant with the co-transport of three Na⁺ [51] and the antiport of one K⁺ along with either one OH⁻ or one HCO³⁻ [41,52]. This process generates a net positive inward current due to excess Na⁺, fueling the transporter [53,54,55].

3. Role of Müller Glial Cells in the Retina

Glial cells support the survival and differentiation of neurons by leading developing neurons and their axons to their targets, constituting an essential component of the blood-brain barrier and contributing to the regulation of extracellular ions and neurotransmitter concentrations [56]. Following the completion of neuronal migration, radial glia cells undergo astrocytic conversion, transforming into multipolar astrocytes [57,58]. Notably, exceptions to this astrocytic conversion occur in the cerebellum and retina, where Bergmann and Müller’s glia persist even into adulthood. MCs are the principal radial glial cells within the inner vertebrate retina, exhibiting a distinct cylindrical, fiber-like morphology, originally termed the ‘Müller radial fibers’ [8].

MCs serve as both anatomical and functional bridges connecting retinal neurons with the compartments essential for molecular exchange, including blood vessels, the vitreous chamber, and the subretinal space. MCs in the retina are highly organized and positioned to enable the performance of a diverse array of physiological functions essential for modulating signaling and impacting the survival of retinal neurons [10]. MCs process enwrap synapses and establish connections with blood vessels. MCs are readily distinguishable by their peculiar morphology, featuring a well-developed endoplasmic reticulum, significant mitochondria observed at the end feet, and varying amounts of glycogen granules [59]. In living retinal preparations, MCs can be labeled by brief exposure to dyes like Mito tracker orange or Cell Tracker green. In culture, MCs nuclei can be distinguished as the largest among various retinal cell types [60]. Nuclei staining is achieved with ethidium bromide, diamidino yellow, or chromomycin A3, which selectively penetrates MCs, displaying a pronounced affinity for nucleic acids [61]. Immunohistochemically, the expression of specific protein markers is confined to astrocytes and MCs, such as vimentin and glial fibrillary acidic protein (GFAP) [62], or selectively to MCs, including the cellular retinaldehyde-binding protein (CRALBP), Glu/Asp transporter (GLAST), and GS [63]. However, MCs have been shown to express GFAP in response to retinal diseases and injury [64].

MCs express a diverse array of receptors, encompassing those for amino acids, catecholamines, neuroactive peptides, hormones, and growth factors [65,66,67]. These receptors in turn exist as co-assemblies of different subunits leading to variations in signal transduction [67]. The activation of receptors in glial cells during neuronal activity suggests the potential involvement of glia in information processing, plasticity, or pathological states [57]. MCs protect the retinal neurons due to their unique properties which involve the secretion of neurotrophic factors, timely Glu uptake, recycling, and the release of antioxidants. MCs contribute to the reduced glutathione pool utilized in neurons, acting as a scavenger for free radicals and reactive oxygen compounds. Specialized high-affinity membrane Glu transporters (EAATs) are vital in preventing neurotoxicity by ensuring the prompt clearance of synaptic glutamate [55]. EAATs are expressed in both neurons and MCs. However, the synaptic responses of RGCs are influenced by the Glu transporters of MCs, as opposed to neuronal transporters. The predominant removal of Glu occurs through uptake by MCs, in which GLAST and Glu transporter 1 (Glt-1) play crucial roles.

Glu metabolism within MCs is connected to its nutritional functions [9]. MCs play a crucial role in generating substrates essential for the oxidative metabolism of photoreceptors and neurons, including but not limited to Gln, lactate, pyruvate, alanine, and α-ketoglutarate [68]. These produced substrates serve as vital energy sources for photoreceptors and neurons during periods of metabolic stress, such as in darkness. Additionally, Glu contributes to forming energy substrates through alternative metabolic pathways, including pyruvate transamination [69]. Within the retina, the predominant site of glucose uptake and metabolism is the inner processes of MCs, specifically localized to the inner plexiform and ganglion cell layers, where glutamatergic signaling is important [70]. Experimental inhibition of glial Glu uptake has demonstrated that even low extracellular Glu concentrations are neurotoxic [71]. Changes in the activity of glial Glu transporters may also contribute to regulating the glial support of neuronal signal transmission from photoreceptors to RGCs [9]. Through their involvement in Glu uptake, MCs contribute to establishing the signal-to-noise ratio of synaptic transmission and the spatial resolution of light-induced signaling [72]. Our lab and others have shown that besides Glu transporters’ role in rapidly removing the amino acid from the synaptic cleft, EAATs also serve as an important signaling molecule involved in neuronal/glia coupling. EAATs are differentially expressed, which strongly suggests that each of these transporters is under a specific transcriptional regulation. For a more detailed description of Glu transporter’s expression, regulation and signaling see these recent reviews [41,73,74,75].

In addition to decreasing extra synaptic Glu levels, thereby preventing spillage to nearby synapses, MCs uniquely harbor glutamine synthetase (GS), an enzyme crucial for converting Glu into Gln [76,77]. The rapid activity of the GS enzyme could be responsible for the resistant properties shown by MCs to excitotoxic Glu levels as described in chick cerebellum-derived Bergmann glia cell (BGC) [78]. However, studies have shown that prolonged exposure to Glu can also induce changes in glial cell morphology without resulting in cell death. Observed alterations include a collapse of the cytoskeleton and a subsequent transition from an epithelioid to a retracted morphology [79].

4. Glu Receptor Classification

Glu receptors fall into two main classes: metabotropic and ionotropic, which are categorized into distinct subtypes and subgroups (Figure 2) based on their affinity to specific Glu analogs, subsequent signal induction mechanisms, sequence homology, and structure [80]. While many Glu receptor agonists and antagonists share structural similarities with Glu, this is not universally true for all compounds. Reviews by Reiner et al. [81] and others [82,83] describe Glu receptors’ pharmacological and molecular properties extensively.

5. Ionotropic Glu Receptor (GRI)

Ionotropic Glu receptors are integral membrane proteins characterized by the assembly of four different subunits (tetramers), each comprising over 900 residues, forming a central ion channel pore [83]. The GRI family are ligand-gated cation channels that account for most of the fast excitatory synaptic transmission in the CNS. It includes α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (GRIA), kainic acid (GRIK), and N-methyl-D-Aspartate receptors (GRIN) [84,85]. GRI are characterized by sequence similarity which suggests a common architectural framework. GRIA and GRIK subunits typically form functional homotetramers or heterotetramers, although NMDA receptors are predominantly heterotetramers [86]. Notably, across all GRI subtypes, the binding sites for pore blockers are consistently present within the transmembrane ion channel, emphasizing a shared pharmacological aspect critical to their function [82]. These receptor subunits adopt a modular structure featuring four distinct domains: the extracellular amino-terminal domain (ATD) which is crucial for subtype-specific receptor assembly, trafficking, and modulation; extracellular ligand-binding domain (LBD) central to agonist/competitive antagonist binding and gating, the domain forming the ion channel (TMD), and an intracellular carboxyl-terminal domain (CTD) domain involved in receptor localization and regulation [84,87]. GRI could undergo RNA editing, a mechanism that serves as a crucial modulator of the permeability properties of these receptors, encompassing the post-transcriptional modification of a particular RNA molecule that changes its coding specificity through processes different from splicing. The most prevalent form of editing is the adenosine-to-inosine (A-to-I) edition, catalyzed by adenosine deaminase acting on RNA (ADAR) I and II [88]. The translational machinery interprets these inosines as guanosines, inducing alterations in the codon of the edited nucleotide and consequent amino acid changes in the protein [89].

6. α-amino-3-hydroxy-5-methyl-4-isoxazole Propionate Receptor (GRIA)

GRIA are fast-acting Glu receptors that conduct signals at a millisecond time frame [83,90]. GRIA are heteromeric complexes composed of four subunits, namely GRIA1, GRIA2, GRIA3, and GRIA4 whose assembly generates receptors permeable to cations with distinct physiological properties dictated by their specific subunit composition [83]. GRIA1–3 mRNAs are ubiquitously expressed throughout the CNS, while GRIA4 mRNA exhibits a more restricted spatial and temporal distribution [91]. Each GRIA subunit has an extracellular N-terminal domain, the LBD features binding sites for modulators influencing receptor desensitization and deactivation, and an intracellular C-terminal tail [90].

The activation of GRIA channels allows the influx of Na⁺ and varying levels of Ca²⁺, depending on the subunits present, inducing excitatory postsynaptic currents (EPSCs) characterized by rapid kinetics and receptor desensitization, influenced by subunit composition [92,93]. Incorporation of GRIA2 limits Ca²⁺ permeability due to Q/R site codon editing within the pore-forming region, with all the GRIA2 subunits undergoing Q/R editing after birth, preventing divalent ion permeation [94,95]. Consequently, receptors lacking the GRIA2 subunit exhibit greater Ca²⁺ permeability. Alternative splicing of all GRIA subunits generates flip and flop variants with differing kinetics of desensitization, with most Flip variants exhibiting slower, less pronounced desensitization, potentially increasing vulnerability to excitotoxic injury if flop variants are decreased, as observed in amyotrophic lateral sclerosis (ALS) [96]. GRIA functions extend beyond neurons to include glial cells, in which the activation of functional GRIA regulates various cellular activities such as cell migration, morphological development, proliferation, differentiation, transcriptional regulation, survival, and ion channel regulation [97,98,99,100].

7. Kainic Acid Receptor (GRIK)

KA receptors are a type of GRI unique due to their presence at post and presynaptic terminals [101]. KA receptors also have different subunits referred to as GRIK1-5, a combination of those subunits forms tetrameric complexes [67]. GRIK comprises two subunit families, categorized by their ligand affinities: low-affinity (GRIK1-GRIK3) and high-affinity (GRIK4-GRIK5). The low-affinity subunits form homomeric or facultative heteromeric channels with both low- and high-affinity subunits. In contrast, high-affinity subunits are obligate heteromers, requiring low-affinity subunits for surface expression.

GRIK is primarily expressed in the CNS, spanning various neuronal types with substantial differences in subunit composition and splice variants across cell types and brain regions. Beyond the CNS, GRIK exhibits differential localization in various tissues, exerting significant regulatory roles in physiological activities under normal and diseased conditions. The expression levels of GRIK are intricately controlled by environmental stimulus, with specific subunits on cells acting as potent determinants of synaptic phenotypes.

The GTEx RNA-seq data highlights the notable expression of GRIK2 and GRIK5 in the cerebellar hemisphere and cerebellum, a finding consistent with supporting evidence from both in vitro and in vivo experiments [102]. Additionally, elevated GRIK5 expression is evident in diverse organs such as the testis, pituitary, colon, sigmoid, thyroid, tibial artery, and uterus, among others. Conversely, GRIK1, GRIK3, and GRIK4 predominantly exhibit expression in brain tissues.

GRIK stimulation exerts crucial modulatory roles in neurotransmission and neuronal development within the mammalian brain, being present on both pre- and post-synaptic sites of neurons [103]. While these receptors function as classical ligand-gated ion channels on postsynaptic neuronal compartments, their involvement in the non-canonical G-protein coupled metabotropic pathway on presynaptic sites allows them to finely regulate the release of excitatory and inhibitory neurotransmitters [104]. This dual modulatory capacity within neuronal circuits positions GRIK as a promising target for therapeutic interventions. Extensive research has utilized GRIKantagonists to mitigate the deleterious effects associated with various neuropathies, including epilepsy, migraine, and pain. Heteromeric GRIK composed of GRIK2 and a high-affinity GRIK subunit, such as GRIK4 or GRIK5, are enhanced by group I GRM activation through a protein kinase C (PKC)-dependent mechanism [105]. This process likely involves the phosphorylation of specific residues in the membrane-proximal C-terminus of GRIK4 or GRIK5 [106]. This regulatory mechanism imparts unique functional characteristics to heteromeric GRIK, potentially serving as a key molecular basis for group I GRM-mediated modulation of GRIK in synaptic plasticity and signal transmission [107].

8. N-methyl-D-Aspartate Receptors (GRIN)

N-methyl-D-Aspartate receptors are the third GRI subtype. These receptors are heterotetramers made up of diverse subunits encoded by three gene families GRIN1, GRIN2, and GRIN3 [108]. Functional GRIN essentially consists of GRIN1 and GRIN2 subunits [109,110]. The specific subunit composition of NMDAR exhibits variations across different synapses and developmental stages. While the GRIN1 subunit is a ubiquitously expressed essential component, GRIN2 subunits (GRIN2A-D) are encoded by distinct genes, displaying differential expression throughout the brain and during development. Notably, GRIN2A and GRIN2B expression patterns are relatively broad, with a concurrent decrease in GRIN2B and an increase in GRIN2A expression as neurons mature. GRIN2C is primarily restricted to the cerebellum and expressed later in development, whereas GRIN2D is predominantly expressed early in development, localized mainly in thalamic and hypothalamic nuclei and the brainstem [111]. The GRIN3A subunit exhibits widespread distribution early in development [112,113], while GRIN3B is primarily confined to motor neurons [114]. Endogenous GRIN typically incorporates GRIN1 and GRIN2 subunits, with GRIN3 subunits playing a modulatory role in the subpopulation of NMDAR [109].

A unique aspect of GRIN is their requisite co-agonist, glycine, in addition to Glu for activation [115]. GRIN subunits consist of a long extracellular N-terminal domain, three true transmembrane segments, a re-entrant pore loop, and an intracellular C-terminal domain of variable length. The C-terminal domain, marked by its considerable divergence among GRIN subunits, plays a pivotal role in introducing diversity to GRIN through different subunit compositions. While the N-terminal domain and extracellular loop form the ligand-binding pocket [116], the C-terminal tail regulates receptor interactions with various cytosolic proteins. These protein–protein interactions dictate the precise intracellular trafficking and localization of NMDA receptors. Additionally, different GRINR subunits are associated with distinct intracellular signaling complexes. For instance, GluN2B specifically interacts with the protein SynGAP, a Ras GTPase activating protein demonstrated to selectively inhibit NMDA-stimulated ERK signaling [117]. Furthermore, GluN2A and GluN2B exhibit differential affinities for active calcium/calmodulin-dependent protein kinase II (CaMKII), resulting in various forms of synaptic plasticity [118,119]. Lastly, the C-termini of GRIN subunits serve as substrates for post-translational modifications, such as phosphorylation. Phosphorylation is a key mechanism regulating channel function and receptor localization at synapses which influences numerous cellular processes, including protein activity, localization, and mobility [120].

In neuronal cells, GRIN channels stand out for several distinctive features, including a voltage-sensitive block by extracellular Mg²⁺, high permeability to Ca²⁺, and remarkably slow activation/deactivation kinetics [108]. The Mg²⁺ block serves as a molecular coincidence switch, with Mg²⁺ removal occurring from the channel pore during postsynaptic cell depolarization. This alleviation of the Mg²⁺ block, coupled with agonist binding, facilitates Ca²⁺ influx through the GRIN channel, subsequently modulating synaptic strength via Ca²⁺-activated signaling cascades [121,122,123]. Retina MCs of chick, rat, and human origin have been reported to express subtypes of NMDAR with distinctive characterization. For example, in chick retina, GRIN has been reported to have a 5-fold lower affinity for glycine and 30-fold lower affinity for D-serine when compared to the NMDAR subtypes in the brain. In addition, the GRIN subunit of the N-terminal and C-terminal splice variants also differ in retinal MCs and neurons [124,125,126].

Glial GRIN possesses a distinctive characteristic that sets them apart from neuronal counterparts—unlike neuronal GRIN, glial cells lack voltage-dependent Mg²⁺ block [127]. This absence of Mg²⁺ block is crucial for the coincidence-detecting function observed [127,128,129]. Reports from astrocytes show that NMDA-evoked currents remain unaffected by extracellular Mg²⁺ [130]. Intriguingly, a similar lack of prominent Mg²⁺ block has been noted in oligodendrocytes, where both NMDA-induced currents and NMDA-triggered _i [Ca²⁺] transients are recorded at physiological Mg²⁺ concentrations [131]. The molecular basis for the low Mg²⁺ sensitivity of glial GRIN remains unexplained; potential explanations include specific expression of GRIN subunits or as-yet-unidentified posttranslational modifications of receptor subunits [132].

9. Metabotropic Glu Receptors (GRM)

Members of each group have a distinct pharmacological profile and can be activated selectively by agonists or allosteric modulators that have no impact on members of the other groups. Ligand binding to the VFD induces a conformational change in the GPCR, activating the G protein, comprised of α, β, and γ subunits in a heterotrimeric complex. In their inactive state, G proteins are associated with guanosine 5’-diphosphate (GDP). Activation prompts the exchange of guanosine 5’-triphosphate (GTP) for GDP within the α subunit. Subsequently, activated G protein subunits modulate various effector molecules, including enzymes, ion channels, and transcription factors. Termination of G protein activity transpires upon hydrolysis of bound GTP to GDP, leading to the reconstitution of the heterotrimer. The C-terminal region of GRM assumes a pivotal role in governing the coupling to the G protein(s), undergoes alternative splicing, and engages in interactions with intracellular signaling molecules and scaffolding proteins [73]. The recognition sequence within the C-terminal tails of both GRM1 and GRM5 facilitates binding with members of the Homer family of anchoring proteins [132,133,134,135,136,137,138]. Subsequently, Homer proteins establish connections through Shank and PSD, forming a scaffolding structure that links to IP₃ receptors, GRIN, and GRIA [139]. This intricate network implies a direct connection between group I GRM and NMDAR, strategically positioning them for the regulation of NMDA receptors at hippocampal synapses [140].

A characteristic shared among G-protein-coupled receptors (GPCRs), including GRM is their capacity to regulate their cell surface density in the presence of continuous agonist stimulation [141]. The prolonged exposure of most GPCRs to their agonists induces a dampening of responsiveness, known as desensitization. Typically, this process involves a combination of mechanisms leading to receptor down-regulation, achieved through reduced receptor synthesis coupled with increased degradation of pre-existing receptors [142]. Furthermore, emerging evidence suggests a less-explored capability wherein chronic agonist exposure results in an up-regulation of the GPCR. This occurs through the recruitment of existing pools of receptors to the plasma membrane, contingent on tissue type and specific conditions [143,144]. These regulatory mechanisms, governing GRM responses to sustained high Glu levels, could play a role in either promoting or mitigating excitotoxicity [145,146].

10. Group I GRM

Group I includes GRM1 and GRM5. These receptors exhibit positive coupling to the inositol trisphosphate (IP₃)-diacylglycerol (DAG) pathway. Upon ligand binding, these receptors engage positively with phospholipase C (PLC) through Gq/11 signaling [147]. Consequently, the increase in intracellular Ca²⁺ activates protein kinase C (PKC). The activation of PKC by group I GRM follows a canonical pathway, in which conventional PKC isotypes are activated by the rise in intracellular Ca²⁺ and most isotypes of PKC require both Ca²⁺ and DAG for optimal activity [148]. Activation of group I GRM leads to the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂), generating IP₃ and DAG. Subsequently, IP₃ induces the release of Ca²⁺ from intracellular stores. Glial cells exhibit the expression of all three types of inositol triphosphate receptors (InsP₃Rs), potentially indicating variability across different brain regions [149]. However, a detailed mapping of InsP₃Rs expression in glial cells from various origins and locations within the brain is still pending. Existing evidence suggests that the activation of InsP₃Rs can elicit Ca²⁺ signals in diverse types of glial cells, observed in both cultured environments and in situ preparations [150].

Group I GRM possesses the characteristic membrane topology of G protein-coupled receptors (GPCRs); an extracellular N-terminus, an intracellular C-terminus (CT), three intracellular loops, and seven transmembrane domains. The intracellular domains of these receptors create a structurally spacious foundation for direct protein–protein interactions, facilitating the binding of various membrane-bound or cytoplasmic proteins [151]. Noteworthy among these binding proteins are protein kinases, including mitogen-activated protein kinases (MAPKs), Src family kinases (SFKs), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and PKC [152,153]. These kinases, localized within the postsynaptic density (PSD) microdomain, bind to the intracellular domains of GRMR1/5 receptors, particularly the CT region, where they phosphorylate specific residues on the receptors [154]. Additionally, scaffolding or adaptor proteins involved in endocytosis and/or exocytosis of membrane-bound receptors engage with GRM1/5. As expected, these dynamic protein–protein interactions have functional consequences, influencing the clustering, trafficking (internalization and externalization), anchoring, and turnover of the receptors [155]. This interplay of protein–protein interactions plays a crucial role in regulating the efficacy of GRM1/5 coupling to various post-receptor signaling pathways and influencing GRM1/5-mediated synaptic plasticity.

Up to this point, several second messenger-dependent protein kinases have been associated with the desensitization of group I GRM. Activation of PKC has been identified in the agonist-induced desensitization of group I GRM [156]. Specifically, PKC-mediated phosphorylation of GRMR1α has been shown to induce receptor desensitization, as reported by Francesconi and Duvoisin in 2000 [157].

Research conducted by multiple groups has proposed that the activation and expression patterns of group I GRM may play regulatory roles in diverse aspects of neurogenesis and synaptogenesis during cortical development [158,159,160,161]. These studies, among others, indicate a strong correlation between the distribution pattern of group I GRM in specific brain regions and their distinct functional roles. Immunocytochemical and electron microscopy investigations suggest that GRM1 and GRM5 are localized in a perisynaptic manner, predominantly within postsynaptic neurons [162]. Beyond the CNS, group I GRM are also found outside the brain, where they fulfill various crucial roles. For instance, they are present in skin cells, contributing significantly to pain sensation. Additionally, these receptors are expressed in osteoblasts, hepatocytes, and heart cells [163].

11. Groups II and III mGluRs

Group II GRM is made up of GRMR2 and GRMR3 [164] while Group III GRM include GRMR4, GRMR6, GRMR7, and GRMR8 [137]. Group II and III receptors are linked to Gαi/o proteins, leading to the inhibition of adenylyl cyclase activity [165]. This inhibition results in a decrease in cAMP formation and protein kinase A activity. Group II/III receptors are either distributed both pre-synaptically and/or post-synaptically [166]. Expression analyses have revealed the presence of GRM4, GRM7, and GRM8 in both the inner nuclear layer (INL) and the ganglion cell layer, suggesting associations with multiple cell types [167]. In contrast, GRMR6 expression has been localized to the INL [168] and the outer plexiform layer (OPL) [169], where bipolar cell somata and dendrites are situated. Immunocytochemically, GRMR6 has been pinpointed to the dendritic tips of ON bipolar cells within the OPL of the rat retina, precisely where they receive input from presynaptic photoreceptor cells [169]. This localization suggests that GRMR6 plays a crucial role in mediating synaptic transmission between photoreceptors and ON bipolar cells, converting the light-induced hyperpolarization of photoreceptors into depolarization of the ON bipolar cells [170]. Glu activation of mGluR6 leads to a Gαo protein-dependent [171] closure of the transient receptor potential melanoma-related 1 (TRPM1) a non-selective cation channel presumably in a constitutively active state and cell hyperpolarization. Gαo2, an alternative splice variant of Gαo, enhances the response sensitivity [172]. A report has, however, implicated Gβγ, rather than Gαo, in the negative regulation of TRPM1 [173]. Additionally, several other proteins are implicated in modulating or scaffolding this cascade, including Gß5, RGS11, RGS7, R9AP, Ret-PCP2, nyctalopin, and potentially Ret-RGS1 [174,175].

Studies in primate retina have revealed that GRM6 aggregates approximately 400 nm away from the release site within the dendritic tips of ON cone bipolar cells, positioning these receptors at a distance comparable to that of OFF cone bipolar dendritic receptors [176,177]. The expression pattern of GRM6 is tightly regulated during development, with widespread expression throughout bipolar soma early in retinal development, followed by an increase in the OPL around the time of eye-opening (day 14), and eventual confinement to the OPL by day 28 [169]. The crucial role of mGluR6 in ON bipolar cells was first demonstrated in mice lacking GRM6, which display a lack of ERG b-wave activity, indicative of ON bipolar cell function. Consistent with this finding, spontaneous mutations in the mouse mGluR6 gene (nob3 and nob4) resulted in negative ERG waveforms and visual behavior abnormalities. Similarly, certain mutations in the human GRMR6 gene lead to autosomal recessive complete congenital stationary night blindness (arCSNB) [178].

Studies have shown that mGluR7 can reduce the activity of NMDAR through an actin-dependent process [179], while agonists targeting mGluR2 and mGluR3 can enhance NMDAR function through kinase-mediated pathways [180]. However, there is a lack of comprehensive research investigating the impact of group II and III mGluR activation on AMPAR/KAR. Recent findings have unveiled a non-standard mechanism of synaptic long-term depression triggered by mGluR3-induced internalization of AMPAR [181]. Group II agonists, Dicarboxycyclopropyl glycine (DGC-IV) and Aminopyrrolidine-2,4-dicarboxylate (APDC) have been shown to possess neuroprotective and antiepileptic effects, respectively [182,183], and reduces cell proliferation in some areas of the brain [184] and could be a therapeutic drug affecting cell apoptosis [185].

12. Distribution, Expression and Regulation of Glu Receptors in Müller Cells

Research investigations using biochemical and molecular methods revealed that Glu receptors present on cerebellum BGCs, and their adjacent Purkinje neurons, possess similar density, features, and properties. In 1992, research by Müller et al. [186] postulated that glial membranes harbor Ca²⁺-permeable GRIA/GRIK that, upon activation, depolarize the cells through the gating of voltage-independent channels [187] and/or electrogenic transport systems [188]. Puro [189] credited the ionotropic receptor activation to the propagation of the phosphoinositide transduction system. Notably, GRI has been identified in retinal MCs and cerebellar BGCs [125,190,191]. The GRIN expression in astrocytes in situ was initially detected by immunocytochemistry and mRNA analysis. The mRNA specific for GRIN1, GRIN2A, and GRIN2B subunits is expressed in cerebellar BGCs [190]. Glu receptor activation induces changes in the repertoire of GRIA/GRK subunits in Bergmann and Müller glial cells. Among the studied subunits, GRIA4 indicates an up-regulation in BGCs at both mRNA and protein levels [57]. Conversely, in MCs, Glu treatment led to a decrease in GRIA4 mRNA and protein expression. These effects are modulated by the group I GRM [57] GRIN1-positive cells were also found in cultured MCs isolated from the adult human retina, as confirmed by a Western blot approach [192].

Glu receptor expression is modulated through phosphorylation. In addition to serine and threonine, tyrosine represents a phosphorylation site crucial for cellular processes. Tyrosine phosphorylation is predominantly catalyzed by tyrosine kinases, encompassing both receptor and non-receptor tyrosine kinases. These enzymes play a pivotal role in modulating the expression and function of proteins by phosphorylating tyrosine residues. Within the scope of Glu receptor regulation, the Src family kinases (SFK) have emerged as a prominent subfamily of non-receptor tyrosine kinases [193]. Among the nine SFK members, five are expressed in the brain [193]. Of particular interest within this subset are Fyn (isoform 1, also known as FynB) and Src, both notably enriched at synaptic sites, suggesting their involvement in targeting local substrates to modulate synaptic transmission and plasticity [194]. Accumulating evidence underscores the roles of Fyn and Src in phosphorylating iGluRs, influencing their physiology [166,195]. Furthermore, recent findings position Fyn as a crucial SFK member in the regulation of group I GRM [193].

The expression of Glu receptors can also be downregulated through the calcium-mediated activation of calpains. Activation of these proteases may lead to the proteolysis of both GRIN and GRIA [196,197,198]. Additionally, the Ca²⁺-dependent activation of calcineurin and calmodulin has the potential to inhibit voltage-gated and NMDAR-gated calcium currents, respectively [199,200].

13. Glu Receptors Localization in Retinal Cells

In recent years, a multitude of studies have employed diverse immunohistochemical and electrophysiological techniques to delineate the distribution of Glu receptors in the vertebrate retina. The elaborated array of receptor subunits and transporter subtypes across various retinal cells is summarized in

Table 1. Expression of Glutamate Receptor subunits in Neuronal and Non-neuronal cells of the Retina. This table shows the expression of diverse Glu receptor subunits in different experimental models.

Cell Type	Identified Receptor Subtypes and Subunits	Organism	Detection Techniques	References
Horizontal Cells	GRIN1, GRIA1, GRIA4, GRIK2	Rat, mouse, rabbit	Immunolabeling, scRNA sequencing	[101,201,202]
Bipolar Cells	GRIN1, GRIN2A, GRIN2B, GRIK1, GRM 4, GRM 6, GRM 7	Rat, rabbit	Immunofluorescence, western blot, immunocytochemistry.	[27,167,203]
Amacrine Cells	GRIN1, GRIN2B, GRIA1, GRIA4, GRIK2, GRM1, GRM2, GRM5, GRMR3	Rat, cat, mouse, chick embryo, Goldfish	Immunolabeling, immunocytochemistry, immunoelectron microscopy	[164,167,204,205]
Retinal Ganglion Cells	GRIA1, GRIA4, GRIK2, GRIN1, GRIN 2A, GRIN2B GRMR1	Mouse, rat	Immunolabeling, immunocytochemistry.	[167,204,206]
Müller Cells	GRIA4, GRIN1	Rat	Immunocytochemistry, Western blot.	[192,206,207,208]

, reflecting the complex components accountable for retinal glutamatergic neurotransmission.

14. Glu Receptor-Mediated Signaling Events in Müller Glial Cells

Radial glial cells exhibit both short-term and long-term responses to neuronal stimulation, involving a diverse array of transcription factors. The interplay of these factors results in the differential expression of genes based on the stimulation of their neurotransmitter receptors [56]. The primary effectors of Glu receptor-mediated signaling on its target cells are Na⁺ and Ca²⁺, which are responsible for the changes in excitability and the triggering of intracellular cascades that mediate glutamatergic transmission [209]. Agonists of both GRI and GRM stimulate Ca²⁺-dependent transcription of various primary response genes, including those from the Fos and Jun families, in astrocytes and cells of the oligodendrocyte lineage [210]. Unlike iGluR signaling, where Glu binding directly activates ion channels, GRM signaling leads to Ca²⁺ influx through indirect ion channel opening and/or second messenger signaling systems [133]. In-depth exploration of intracellular signaling pathways activated by mGluR has been conducted extensively in neuronal cells, yet there is a comparatively limited understanding of glial cells [211].

In 1994, Sanchez and Ortega reported that in BGC, the activation of the AMPAR/KAR subtype increased the DNA binding activity of the activator protein 1 (AP-1) family of transcription factors in a dose- and time-dependent manner [212]. Alterations in intracellular Ca²⁺ concentration resulting from the activation of Glu receptors can influence gene transcription in glial cells by modulating the expression and activity of transcription factors [213]. Although the activity of Glu transporters (GLAST/EAAT1) on the membrane of the glial cells also contributes to the concurrent rise in intracellular Na⁺ levels, and the subsequent reverse operation of the Na⁺/Ca²⁺ exchanger (NCX), leading to an elevation in intracellular Ca²⁺ concentration, has emerged as a pivotal signaling mechanism in astrocytes [214]. For instance, in BGC, the downstream cascades differ; the receptor-mediated cascade leads to a PKC-mediated transcriptional response [215] and a Ca²⁺/calmodulin-mediated inhibition of protein synthesis elongation [216]. In contrast, Glu transporter-triggered Ca²⁺ influx is predominantly linked to translational control via the activation of the mechanistic target of rapamycin (mTOR) [217].

The activation of Glu-induced Ca²⁺ entry leads to the activation of multiple kinases including Protein Kinase C (PKC) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) [218,219]. Calmodulin (CaM) is a pivotal calcium-binding protein involved in numerous cellular physiological processes [220]. Comprising two globular lobes, CaM exhibits the capability to bind two Ca²⁺ individually [221]. The complexity of PKC signaling is underscored by molecular cloning and sequencing, revealing a diverse family of PKC enzymes. The classical subtypes (alpha, beta I, beta II, and gamma) are activated by phorbol esters and Ca²⁺; the novel subtypes (delta, epsilon, eta, and theta) are also activated by phorbol esters but are Ca²⁺-independent; and the atypical isoforms (zeta and lambda) are not activated by phorbol esters [222]. PKCs, respond differently to cofactor dependence. Notably, PKCα and PKCε participate in the transcriptional regulation of GLAST [215]. This same group also reported that the activated GRIA subtype undergoes tyrosine phosphorylation, through a Src-dependent mechanism providing docking sites for transduction molecules like PI3K [223]. Among the signal transducers activated by phosphorylated inositides is PKC. Also, in BGC, GRIA activation is connected to the transcriptional regulation of chick kainate binding protein (chKBP) in a biphasic manner, mediated by AP-1 and Oct-2 [215,219,224,225]. In another contribution, this same group delved into the interaction between chick cerebellar GRIA and signaling proteins such as phosphorylated focal adhesion kinase (pp125FAK), PI-3K, and paxillin, both in cultured BGCs and cerebellar slices [223,226]. The dynamic interaction between transcription factors in BGC may contribute to establishing, maintaining, or inhibiting neuron-glia interactions, particularly in the context of Ca²⁺-permeable GRIA [99]. In a subsequent study, this same group showed that Glu and AMPA also increase the interaction of the transcription factor Ying Yang 1 (YY1) with the GLAST promoter, and overexpression of this transcription factor decreases GLAST expression [227]. These studies suggest that the effects of AMPA may depend on both AP-1 and YY1. Using the same model, Poblete-Naredo and her colleagues demonstrated that insulin increases YY1 binding to the GLAST promoter by EMSA decreasing GLAST expression [228].

Glu has been shown to modify gene expression in MCs through GRIN-mediated activation of transcription factor pathways and to evoke the release of neuroactive compounds that feedback to neurons [125,229,230]. Among these compounds, studies have identified D-serine, which is released upon Glu stimulation, mimicking the activity of glycine at the strychnine-insensitive site of GRIN, and could act as an endogenous Glu co-agonist. In the vertebrate retina, D-serine is present in MCs and astrocytes and has also been shown to contribute to the physiological activation of GRIN in retinal neurons [231]. Our findings demonstrate that selective activation of GRIN enhances [³H]-D-aspartate uptake, suggesting an increase in the abundance of GLAST/EAAT1 transporters at the plasma membrane [232]. Additionally, treatment with NMDA leads to the association of the transporter with ezrin and GFAP proteins, known to play roles in retaining GLAST at the plasma membrane [232].

Group I mGluR is known to activate MAPK, a pivotal player in protein synthesis-dependent neuronal plasticity [211,233]. The translation and transcription factors modulated by MAPK cascades following the activation of GRM have been thoroughly characterized [211]. In glial cells, particularly astrocytes, a more comprehensive analysis reveals that stimulation of MAPK and PI3K pathways via GRM3 enhances the production of neurotrophic factors, promoting neuroprotection against various toxic insults [234,235,236]. In Group III GRM, the activation of GRM4 receptors in cultured rat neural stem cells leads to the inhibition of JNK and p38 mitogen-activated protein kinase. This downregulates the expression of procaspase-8/9/3 and reverses the Bcl-2/Bax balance, ultimately preventing H₂O₂-mediated cell death [237]. Additionally, a protective role for mGluR7 has recently been discovered in glial cells, involving the activation of PI3K/Akt and MAPK-ERK1/2 pathways [238]. Activation of group II GRM up-regulates GLAST mRNA and protein levels [239,240] via the extracellular signal-regulated kinase (ERK)/PI3K/NF-κB pathway [241]. A summary of these findings is presented in Figure 3.

15. Impact of Glu Receptor Signaling in Excitotoxicity and Retinal Degeneration

The visual system is susceptible to a myriad of pathophysiological conditions collectively referred to as retinal degenerative diseases characterized by distinct clinical pathology and features [242,243,244]. The progressive accumulation of Glu in the extracellular space and over-activation of neuronal Glu receptors has been linked as one of the main causes of retinal diseases. Glu possesses potent excitotoxic properties towards neuronal cells and exposure to micromolar concentrations can lead to both immediate and delayed cell death [245,246]. In cultured retinal neurons, the activation of GRIN and the subsequent influx of Ca²⁺ are considered the primary mechanisms underlying Glu neurotoxicity [177,247]. Excessive extracellular Glu concentration in RGCs leads to uncontrolled continuous depolarization, termed excitotoxicity [248]. Excitotoxicity is characterized by neuronal death due to prolonged Glu exposure and GRIA and GRIN activation not only at the synaptic cleft, but importantly, the activation of extra synaptic GRIN receptors, with a differential subunit composition (enrichment of highly Ca²⁺ permeable GRIN2B subunits) and the consequent excessive Ca²⁺ influx that activates enzymes responsible for the degradation of proteins, membranes, and nucleic acids [249], [250]. GRMs contribute as well, to the increase in intracellular Ca²⁺ concentrations [251]. In the brain, the activation of GRIA and GRIN are involved in synaptic plasticity, culminating in the induction of long-term potentiation (LTP), a sustained enhancement in signal transmission [252,253]. These receptors facilitate the influx of Ca²⁺ into the cell, triggering various intracellular signaling pathways, receptor trafficking, gene expression, and the generation of LTP.

Glu receptor subunit composition and expression could determine the influx of ions, particularly the GRIA2 subunit with widespread expression patterns [254]. While most neurons containing GRIA express GRIA2, there are variations in the expression levels. In numerous brain regions, approximately 8–15% of neurons express Ca²⁺-permeable GRIA lacking GRIA2, and these specific neuronal subpopulations are selectively eliminated in a Ca²⁺-dependent manner when exposed to either AMPA or KA [66,243]. The presence of low but persistent permeability to Ca²⁺ in heterogeneous cell populations might elucidate why mammalian neurons are highly susceptible to GRIA-mediated damage, such as hippocampal granule and pyramidal neurons [255] as well as cortical neurons [256], given the fact that these cells still express GRIA2. However, it remains uncertain whether low Ca²⁺ permeability is the sole factor responsible for GRIA-mediated excitotoxic cell death, although evidence from experiments involving mice lacking a functional GRIA2 gene suggests that increased Ca²⁺ influx in CA1 neurons does not lead to neuropathological lesions indicative of excitotoxicity [257,258]. Furthermore, in MCs, activation of nuclear factor kappa B (NF-kB) leads to increased surface levels of Ca²⁺-permeable GRIA in RGCs, triggering cell death [259].

The intracellular accumulation of Ca²⁺ serves as the trigger for activating protein kinases and other downstream Ca²⁺-dependent enzymes, ultimately leading to cell death through molecular modifications such as cellular membrane damage, the release of reactive oxygen species (ROS), mitochondrial dysfunction, upregulation of neuronal nitric oxide synthase (nNOS), endoplasmic reticulum (ER) stress, and release of lysosomal enzymes, culminating in excitotoxic, oxidative, and apoptotic stress [260,261,262]. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is a protein kinase subject to regulation by the Ca²⁺/calmodulin complex [263]. In certain neuronal disorders such as stroke, epilepsy, and glaucoma, increased Ca²⁺ levels may dysregulate CaMKII activity [264,265,266]. In preventing neuronal damage both in vitro and in vivo, CaMKII inhibition before excitotoxic injury emerges as a potential therapeutic strategy [267]. This emphasizes the significance of understanding the role of CaM and CaMKII in the context of Ca²⁺ regulation, offering valuable insights into the pathophysiology of various neurological conditions [268].

GRIA featuring Q/R-unedited GRIA2 subunits also exhibit permeability to zinc, potentially implicating zinc in processes related to differentiation [269,270]. Studies have demonstrated that astrocytes with mutant superoxide dismutase type 1 (SOD1) release factors that decrease the expression of this specific subunit in motor neurons. This, in turn, results in AMPAR-mediated excitotoxicity and eventual cell death [271,272]. There is experimental evidence indicating that the excessive activation of Ca²⁺-permeable GRIA containing GRIA4 is linked to a death signaling pathway, which is influenced, at least in part, by the AP-1 transcription factor [273]. Furthermore, in the rat retina, GRM activation has been shown to mitigate the detrimental impact of NMDA [274]. Additionally, neuronal injuries give rise to adaptive alterations in GRM expression reminiscent of developmental patterns. During development, RGCs express transcripts for various types of mGluR, some of which decrease in expression during adulthood. However, following neuronal injury, this developmental downregulation appears to be reversed [177].

The impact of elevated levels of extracellular Glu is exacerbated when the activity of the Glu uptake/transporter system is compromised [275,276,277,278]. Dysfunction in the Glu transport into MCs contributes to an increase in extracellular Glu, reaching excitotoxic levels. Research evidence has demonstrated the vulnerability of astrocytic Gln synthetase to inactivation induced by reactive oxygen species (ROS), disrupting the entire Glu/Gln cycle and contributing to an elevation in extracellular Glu concentration, thereby promoting excitotoxicity [279]. Furthermore, the presence of ROS has been observed to diminish Glu transporter activity, hindering the synaptic clearance of Glu and further amplifying the rise in extracellular Glu concentration [53]. Oxygen deprivation can also trigger excitotoxicity through a rather distinct mechanism. Directly, hypoxic-ischemic conditions stimulate Glu release, escalating the extracellular Glu concentration to neurotoxic levels in a straightforward manner [280]. Additionally, oxygen deprivation induces energetic stress by disrupting mitochondrial oxidative phosphorylation, thereby impeding ATP production [281]. This disruption results in intracellular ATP depletion, preventing the reuptake of Glu resulting in an excitotoxic Glu extracellular concentration [282]. The main utilizers of ATP in the brain are ion pumps responsible for sustaining ion gradients crucial for neuronal signaling and communication [283,284,285]. The effect of excessive Glu release leads to the uncontrolled stimulation of Glu receptors, which leads to the massive influx of Ca²⁺ into postsynaptic neurons due to low ATP levels, hence impairing ion pump function [286,287].

16. Role of Müller Glial Cells in Retinal Diseases Prevention

In retinal degeneration, MC physiology has been studied to detect early signs of retinal neuronal death. Studies have shown that early or detectable stages of retinal degeneration are characterized by alterations of important features of MCs such as an increased expression of glial fibrillary acidic protein (GFAP), cellular retinaldehyde-binding protein (CRALBP) and Gln synthetase, undergoing varying degrees of loss [288]. In the later stages of retinal diseases, the metabolic profile and protein expression of Müller cells exhibit a distinctive “chaotic” pattern [289].

The inhibition of Gln synthetase in MCs through pharmacological blockade leads to a reduction in the Glu levels within bipolar and ganglion cells, resulting in functional blindness [290]. Gln synthetase downregulation contributes to neuronal dysfunction and exacerbates Glu toxicity in diverse retinopathies [291]. Glu accumulation inhibits cystine uptake, reinforcing the event by depleting cysteine and glutathione’s reducing potential. Glu-induced nitric oxide (NO) generation and subsequent interactions with oxygen radicals contribute to delayed death of retinal neurons in retinal ischemia, further accelerating RGC death.

Understanding the cellular mechanisms involved in the impairment of glial Glu recycling may help to find novel targets for developing neuroprotective agents. An increase in Gln synthetase in MCs may represent an attractive approach that has been shown to protect against neuronal degeneration in the injured retinal tissue [292]. The decrease in uptake might be caused by the downregulation/inactivation of Gln synthetase, by mitochondria-derived free radicals, which inactivate the transporter molecules, as well as by cell depolarization resulting from high extracellular K⁺, energy failure due to mitochondrial dysfunction, downregulation/inactivation of K⁺ channels, and inhibition of Na^*/K⁺ ATPase and Glt-1 through inflammatory lipid mediators [291].

Prolonged exposure to Glu can induce alterations in the morphological features of glial cells without necessarily leading to cell death. When retinal MCs are exposed to Glu, the cytoskeleton undergoes a collapse, resulting in a transition from an epithelioid to a retracted morphology [187]. These morphological modifications might potentially impact the ability of MCs to regulate extracellular K⁺ levels released during neuronal activity [79] and the expression of neurotransmitter receptors and transporters situated in the membrane.

Transient retinal ischemia or diabetes does not significantly alter GLAST expression or its evoked membrane currents, it reduces the efficiency of Glu transport into MCs. This leads to a high Glu influx into photoreceptor, bipolar, and ganglion cells under these conditions. In patients with glaucoma, a downregulation of GLAST (but not Glt-1) has been observed [276]. Increased intraocular pressure in experimental glaucoma results in a failure of GLAST activity, leading to decreased Glu accumulation in MCs and significant Glu uptake by RGCs, coinciding with excitotoxic damage to the retina [243]. The elevated intraocular pressure causes inner retinal hypoxia, triggering the formation of free radicals, and disrupting Glu transport in MCs. Similar dysfunction in electrogenic Glu transport caused by oxidative stress is observed in experimental diabetes [293].

This malfunction in Glu transport into MCs, induced by free radicals from mitochondria, may explain retinal ganglion cell death in Leber hereditary optic neuropathy [294]. MCs from patients with retinopathies display increased density of Glu transporter currents, and experimental retinal detachment shows an increase in GLAST labeling. However, under these conditions, MCs depolarize due to functional inactivation or downregulation of inwardly rectifying K⁺ channels (Kir) [295]. Various retinal diseases associated with a breakdown of blood-retinal barriers result in serum leakage into the retina, impacting Glu transport efficiency. Clinical observations link hemorrhages at vascular leakage sites with a greater reduction in retinal function, possibly linked to the decreased efficiency of Glu transport into MCs [296]. The depolarization of MCs may be strong enough to reverse the operation mode of Glt-1 resulting in an efflux of Glu that contributes to excitotoxic neuronal damage. A significant portion of glial Glu might also be released through the cystine-glutamate antiporter and vesicular exocytosis [69].

Above we describe the signaling pathways and how these could be activated by Glu receptors. These pathways have an important role in different organisms. Diverse studies have demonstrated the activation of phosphoinositide (PI) hydrolysis in MCs which activate the PKC pathway [230]. The PKC family might be involved in regulating synaptic actions in glutamatergic systems such as blood-brain barrier homeostasis, apoptotic cycle, immune regulation, signal transduction, and transmission which allows the development and differentiation of tissues and cells; those activities give these enzymes an important role in physiology [297,298]. Activation of PKC is also involved in the PLC pathway which is characterized to activate and generate the second messengers DAG and PIP₂. Generally, PLC allows lipidic hydrolysis and second messengers generation, and that leads to cell regulation by proliferation and migration; PLC regulates immune responses, apoptotic cycle, and neuronal signaling [299]. Due to their involvement in these important processes, dysfunction in the pathway most possibly leads to disease. In retina illnesses such as Diabetic retinopathy, resulting from damaged retinal blood vessels, it could lead to permanent blindness. An increase in PKC activity has been reported in the generation of new blood vessels, a process known as neovascularization [300,301].

Several diseases characterized by reduced Glu transporter expression are linked to alterations in DNA methylation patterns. Recently, our group reported the effect of Glu on global 5-methylcytosine (5-mc) content in radial glial cells, this study implicates dynamic DNA methylation/demethylation in gene expression regulation of glial Glu transporters [302]. Retinal disorders such as age-related macular degeneration and diabetic retinopathy are linked to dynamic DNA methylation [303]. For instance, hypermethylation of the Glt-1 promoter is observed in brain tumors [304]. Aberrant methylation control underlies Rett syndrome, a neurodevelopmental disorder resulting from mutations in the DNMT methyl-CpG-binding protein 2 (MeCP2), resulting in dysregulation of both GLAST and Glt-1 [305,306,307].

An essential progression from these molecular investigations and a future research challenge, involves identifying downstream genes regulated by transcription factors associated with Glu-receptor-mediated signaling in glia. For instance, some clinical studies reported that many Glu agonists and antagonists exhibit neuroprotective effects in retinal neuronal cells, and t-ACPD, a mGluR Group I and II agonist, possesses neuroprotective effects against ischemic damage [308]. Also, the mGluR1 antagonist, CPCCOEt could possess neuroprotective abilities against excitotoxic-induced death [309], similarly, an agonist of mGluR group II, like LY354740 has a neuroprotective effect that is enhanced by glial cells [310], in addition to that, in the neuroprotective effect of Group II, it is believed that transforming growth factor- β (TGFβ) is the main intermediary [311]. Likewise, a GRI antagonist, such as ifenprodil, could produce, like the other agonists and antagonists, a neuroprotective effect without modifying the synaptic transmission [312]. The Group II agonist LY354740 reverses RGC hyperexcitability and increases the expression level of Brain-derived neurotrophic factor (BDNF), thus providing a neuroprotective effect [313].

17. Perspectives

The evidence reviewed above strongly suggests a complex functional interaction between retinal neurons and MCs hence, influencing their information processing and behavioral output. Glu receptors modulating glutamatergic neurotransmission are expressed on both neuronal and glial cells, this similarity makes it complicated to modulate their downstream activities as this would lead to various side effects that might complicate the assessment of therapeutic efficacy. However, a promising alternative could be exploring glial-cell-specific downstream molecular targets which present an avenue to develop optimized therapeutics. Current evidence is limited to in vitro and brain slice experiments, which limit our understanding of the functional role of all these components in vivo.

This review highlights the relevance of Glu receptor activation on glial-neuronal communication in the retina. Arising questions are still to be discussed. What influences the differential expression patterns of Glu receptors? How does Glu receptor activation affect visual functions at molecular and physiological levels? What are the different regulatory mechanisms through Glu receptor signaling involved in the expression of Glu transporters? How do epigenetic modifications, such as DNA methylation and histone modifications, regulate the expression of Glu receptors and transporters? What signaling pathways and transcription factors are involved in the regulation of Glu transporters, and how do they contribute to neurological disorders?

18. Conclusions

We focused on Glu receptor-mediated signaling in MCs due to their significant role in modulating glutamatergic transmission in the retina. For many years, there has been a paucity of adequate information on the role of MC signaling in ocular diseases, but recent research has made it clear that glial cells are indispensable for understanding the mechanism of retinal diseases. In the future, a large effort will be required for the detailed localization of the different classes of GRM and GRI and their various subunits on the MCs, because the functional properties of receptors depend on the subunit composition, it will be especially important to determine which receptor subunits are expressed at the synapse during the light/dark cycle. Still, further research is needed to bridge the substantial gaps in our understanding of mechanisms and physiological relevance of glial-neuron glutamatergic interactions, in particular, the impact of glial Glu receptor activation and its emerging roles in retinal degeneration and diseases.