Müller cell swelling, glutamate uptake, and excitotoxic neurodegeneration in the isolated rat retina

We characterized morphological effects of the endogenous excitotoxin, glutamate in ex vivo retinal segments prepared from 30-day-old rats. Initial changes induced by glutamate consisted of reversible, sodium-dependent Müller cell swelling. This glial swelling was mimicked by glutamate transport substrates but not by ionotropic glutamate receptor agonists. Only very high concentrations of exogenous glutamate (3,000 microM) produced excitotoxic neuronal damage. The neuronal damage was accompanied by severe glial swelling and was blocked by an antagonist of non-N-methyl-D-aspartate (NMDA) receptors but not by an NMDA receptor antagonist. Because glutamate uptake can be influenced by changes in cellular energy levels, we studied the effects of oxidative and glycolytic energy depletion on glutamate-mediated Müller cell swelling. Oxygen deprivation produced little morphological change and did not alter either glutamate-mediated Müller cell swelling or glutamate-induced excitotoxicity. In contrast, inhibition of glycolysis by iodoacetate produced severe neuronal damage without Müller cell swelling. In the presence of iodoacetate, exogenous glutamate failed to cause glial swelling. The neuronal damage produced by iodoacetate was inhibited by pyruvate, a substrate that sustains oxidative energy pathways. In the presence of iodoacetate plus pyruvate, glutamate failed to cause Müller cell swelling but became neurotoxic at low concentrations through activation of non-NMDA receptors. These results indicate that glycolytic energy metabolism plays a critical role in sustaining ionic balances required for Müller cell glutamate uptake and glial uptake helps to prevent glutamate-mediated excitotoxicity.     

Role of ammonia in reversal of glutamate-mediated Müller cell swelling in the rat retina

Glutamate is thought to participate in a variety of retinal degenerative disorders. However, when exposed to glutamate at concentrations up to 1 mM, ex vivo rat retinas typically exhibit Müller cell swelling, but not excitotoxic neuronal damage. This Müller cell swelling is reversible following glutamate washout, indicating that the glial edema is not required for glutamate-induced neuronal injury. It is unclear whether glutamate directly induces the Müller cell swelling or whether a metabolite of glutamate such as glutamine acts as an osmolyte to generate the cellular edema. To examine this issue, ex vivo rat retinas were exposed to 1 mM glutamate or 1 mM glutamine and were evaluated histologically. Glutamate was also combined with 1 mM ammonia or with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase, the enzyme that catalyzes the synthesis of glutamine from glutamate and ammonia. Glutamate-mediated Müller cell swelling was blocked by co-administration of ammonia and the reversibility of Müller cell swelling was inhibited by MSO administered following glutamate exposure. Glutamine alone failed to induce Müller cell swelling. These results indicate that glutamate-mediated Müller cell swelling is unlikely to result from glutamine accumulation. Rather, conversion of glutamate to glutamine in a reaction involving ammonia helps reverse Müller cell swelling following exposure to exogenous glutamate.     

New functions of Müller cells

Müller cells, the major type of glial cells in the retina, are responsible for the homeostatic and metabolic support of retinal neurons. By mediating transcellular ion, water, and bicarbonate transport, Müller cells control the composition of the extracellular space fluid. Müller cells provide trophic and anti‐oxidative support of photoreceptors and neurons and regulate the tightness of the blood‐retinal barrier. By the uptake of glutamate, Müller cells are more directly involved in the regulation of the synaptic activity in the inner retina. This review gives a survey of recently discoved new functions of Müller cells. Müller cells are living optical fibers that guide light through the inner retinal tissue. Thereby they enhance the signal/noise ratio by minimizing intraretinal light scattering and conserve the spatial distribution of light patterns in the propagating image. Müller cells act as soft, compliant embedding for neurons, protecting them in case of mechanical trauma, and also as soft substrate required for neurite growth and neuronal plasticity. Müller cells release neuroactive signaling molecules which modulate neuronal activity, are implicated in the mediation of neurovascular coupling, and mediate the homeostasis of the extracellular space volume under hypoosmotic conditions which are a characteristic of intense neuronal activity. Under pathological conditions, a subset of Müller cells may differentiate to neural progenitor/stem cells which regenerate lost photoreceptors and neurons. Increasing knowledge of Müller cell function and responses in the normal and diseased retina will have great impact for the development of new therapeutic approaches for retinal diseases.     

Immunocytochemical localization of the glutamate transporter GLT-1 in goldfish (Carassius auratus) retina

Glutamate is the major excitatory neurotransmitter in the retina of vertebrates. Electrophysiological experiments in goldfish and salamander have shown that neuronal glutamate transporters play an important role in the clearance of glutamate from cone synaptic clefts. In this study, the localization of the glutamate transporter GLT-1 has been investigated immunocytochemically at the light and electron microscopical levels in the goldfish retina using a GLT-1-specific antibody. GLT immunoreactivity (IR) was observed at the light microscopical level in Müller cells, bipolar cells, the outer plexiform layer (OPL), and the inner plexiform layer (IPL). At the electron microscopical level, membrane-bound and cytoplasmic GLT-IR in the OPL was located in finger-like protrusions of the cone terminal located near the invaginating postsynaptic processes of bipolar and horizontal cells. GLT-IR was not observed in the vicinity of synaptic ribbons. This location of GLT-1 allows modulation of the glutamate concentration in the synaptic cleft, thereby shaping the dynamics of synaptic transmission between cones and second-order neurons. In the inner IPL, GLT-IR was observed in the cytoplasm and was membrane bound in mixed rod/cone bipolar cell terminals and cone bipolar cell terminals. The membrane-bound GLT-1 was generally observed at some distance from the synaptic ribbon. The morphology of the bipolar cell terminal together with the localization of GLT-1 suggests that at least these glutamate transporters are not primarily involved in rapid uptake of glutamate release by the bipolar cells. The GLT-IR in the cytoplasm of Müller cells was located throughout the entire goldfish retina from the outer limiting membrane to the inner limiting membrane. The location of GLT-1 in Müller cells is consistent with the role of Müller cells in converting glutamate to glutamine.     

Glutamate transporter EAAC1 is expressed on Müller cells of lower vertebrate retinas

The expression of glutamate transporter EAAC1 was investigated in carp and bullfrog retinas using Western blotting, immunofluorescence double labeling and confocal laser scanning microscopic techniques. In addition to a variety of retinal neurons, radially oriented elements spanning the whole neural retinas of carp and bullfrog were also EAAC1-immunoreactive, and EAAC1 was found to be predominantly on the cell membrane. Virtually all EAAC1-labeled radial elements were immunopositive to glial fibrillary acidic protein (GFAP), a specific marker for retinal Müller cells of carp and bullfrog, indicating that they were Müller cells. This finding suggests that EAAC1, which has been thought to be an exclusively neuronal type, may be a glial transporter as well. EAAC1 of Müller cells may play an important modulatory role in the retina by making contributions to glutamate homeostasis.     

Comparative electrophysiology of retinal Müller glial cells—A survey on vertebrate species

Müller cells are the dominant macroglial cells in the retina of all vertebrates. They fulfill a variety of functions important for retinal physiology, among them spatial buffering of K⁺ ions and uptake of glutamate and other neurotransmitters. To this end, Müller cells express inwardly rectifying K⁺ channels and electrogenic glutamate transporters. Moreover, a lot of voltage‐ and ligand‐gated ion channels, aquaporin water channels, and electrogenic transporters are expressed in Müller cells, some of them in a species‐specific manner. For example, voltage‐dependent Na⁺ channels are found exclusively in some but not all mammalian species. Whereas a lot of data exist from amphibians and mammals, the results from other vertebrates are sparse. It is the aim of this review to present a survey on Müller cell electrophysiology covering all classes of vertebrates. The focus is on functional studies, mainly performed using the whole‐cell patch‐clamp technique. However, data about the expression of membrane channels and transporters from immunohistochemistry are also included. Possible functional roles of membrane channels and transporters are discussed. Obviously, electrophysiological properties involved in the main functions of Müller cells developed early in vertebrate evolution. GLIA 2017;65:533–568     

Glutamate stimulates neurotrophin expression in cultured Müller cells

The uptake of excess extracellular glutamate and the secretion of neurotrophins by glial cells have been suggested to protect CNS neurons from glutamate-induced toxicity. In the retina, perturbation of glutamate transport and decreased retrograde transport of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) may contribute to ganglion cell death in experimental glaucoma. Although many studies show a clear relationship between glutamate and neurotrophic factors, such relationship has not been thoroughly investigated in the retinal environment. In the following study, we determined the effects of glutamate on early passaged rat Müller cells, specifically their expression of neurotrophic factors including BDNF, nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and glial-cell line derived neurotrophic factor (GDNF); and of glutamate receptors and transporters using immunoblots or enzyme-linked immunosorbent assays. Binding of BDNF to its cognate receptor TrkB was also determined using co-immunoprecipitation studies. Cultured Müller cells grown in the presence of glutamate were also assayed for survival using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). Our study showed that while glutamate treatment did not promote cell death, it upregulated secretion of BDNF, NGF, NT-3, NT-4, and GDNF by Müller cells. While solitary bands at approximately 13-14 kDa were observed for NGF, NT-3, and NT-4; two BDNF-reactive bands were observed in immunoblots: a faster migrating band at the reported size of the BDNF monomer (approximately 13 kDa); and a more intense band at approximately 36 kDa. GDNF-reactive bands were observed at approximately 22, approximately 28, and approximately 55 kDa. Glutamate also induced significant changes in glutamate receptor and transporter proteins, as well maintained the association of BDNF to TrkB in Müller cells. The decreased N-methyl-D-aspartate receptor (NMDAR) levels and sustained activation of TrkB by BDNF could serve as protective mechanisms for Müller cell survival. Moreover, the increased secretion of neurotrophic factors and upregulation of L-glutamate/L-aspartate transporter (GLAST) expression in Müller cells may protect retinal neurons from glutamate toxicity.     

Calcium signaling in specialized glial cells

This article reviews calcium signaling in three specialized types of glial cells: Müller cells of the retina, Bergmann glial cells of the cerebellum, and radial glial cells of the developing cortex. Müller cells generate spontaneous and neuronal activity-evoked increases in Ca(2+). Neuron to Müller cell signaling is mediated by neuronal release of ATP and activation of glial P2Y receptors. Müller cells, in turn, modulate neuronal excitability and mediate vasomotor responses. Bergmann glial cells also generate spontaneous and activity-evoked Ca(2+) increases. Neuron to Bergmann glia signaling is mediated by neuronal release of nitric oxide, noradrenaline, and glutamate. In Bergmann glia, Ca(2+) increases control the structural and functional interactions between these cells and Purkinje cell synapses. In the ventricular zone of the developing cortex, radial glial cells generate spontaneous Ca(2+) increases that propagate as Ca(2+) waves through clusters of neighboring glial cells. These Ca(2+) increases control cell proliferation and neurogenesis.     

Glutamate release by neurons evokes a purinergic inhibitory mechanism of osmotic glial cell swelling in the rat retina: activation by neuropeptide Y

Glial cell swelling is a central cause of ischemic edema in the brain and retina; however, the regulation of glial cell volume by endogenous factors in situ is largely unknown. In slices of the postischemic retina of the rat, the somata of glial (Müller) cells swell upon hypotonic stress that is not observed in slices of control retinas. We describe an endogenous signaling pathway that leads to inhibition of the osmotic glial cell swelling, and that is evoked by the release of glutamate from retinal neurons upon application of neuropeptide Y. Glutamate activates metabotropic glutamate receptors on swollen glial cells, which evokes a Ca2+ -independent purinergic signaling cascade that involves release of ATP, P2Y1 receptor activation, and transporter-mediated release of adenosine. Activation of A1 receptors causes the inhibition of osmotic glial cell swelling, by a protein kinase A-dependent activation of K+ and Cl- channels. It is proposed that the glutamate-evoked purinergic receptor signaling of glial cells is crucially involved in the cell volume homeostasis of the retina, and that this mechanism may contribute to the protective effect of adenosine in the ischemic tissue.     

Selective staining by vital dyes of Müller glial cells in retinal wholemounts

Müller glial cells within the retina may respond to different signaling molecules with an elevation of their intracellular free calcium. To prove the localization of the recorded calcium responses in Müller cells within acutely isolated retinal wholemounts, retinal pieces from adult animals and humans were exposed to different vital dyes just after the calcium imaging records were finished. The dyes, Mitotracker Orange, Mitotracker Green, Celltracker Orange, Celltracker Green, and monochlorobimane, are all selectively taken up by Müller glial cells, while neuronal cells remain largely devoid of the dyes. By using this method, it can be demonstrated that the free calcium alterations within the wholemounts indeed occur within Müller cells. Moreover, the cross-sectional areas of (dye-filled) Müller glial cell bodies, as well as of (dye-free) neuronal cell bodies, can be measured in retinal wholemounts, and the spatial densities of both types of cells can be determined. The vital dye loading of Müller cells may facilitate investigations of stimulus-induced alterations of retinal glial cell physiology and morphology.     

Two different mechanosensitive calcium responses in Müller glial cells of the guinea pig retina: Differential dependence on purinergic receptor signaling

Tractional forces or mechanical stimulation are known to induce calcium responses in retinal glial cells. The aim of the study was to determine the characteristics of calcium responses in Müller glial cells of the avascular guinea pig retina induced by focal mechanical stimulation. Freshly isolated retinal wholemounts were loaded with Mitotracker Deep Red (to fill Müller cells) and the calcium‐sensitive dye Fluo‐4/AM. The inner retinal surface was mechanically stimulated with a micropipette tip for 10 ms. Stimulation induced two different cytosolic calcium responses in Müller cells with different kinetics in dependence on the distance from the stimulation site. Müller cells near the stimulation site displayed an immediate and long‐lasting calcium response with high amplitude. This response was mediated by calcium influx from the extracellular space likely triggered by activation of ATP‐insensitive P2 receptors. More distant Müller cells displayed, with a delay of 2.4 s, transient calcium responses which propagated laterally in a wave‐like fashion. Propagating calcium waves were induced by a calcium‐independent release of ATP from Müller cells near the stimulation site, and were mediated by a release of calcium from internal stores triggered by ATP, acting in part at P2Y₁ receptors. The data suggest that mechanically stimulated Müller cells of the guinea pig retina release ATP which induces a propagating calcium wave in surrounding Müller cells. Propagating calcium waves may be implicated in the spatial regulation of the neuronal activity and homeostatic glial functions, and may transmit gliosis‐inducing signals across the retina. Mechanical stimulation of guinea pig Müller cells induces two calcium responses: an immediate response around the stimulation site and propagating calcium waves. Both responses are differentially mediated by activation of purinergic receptors. GLIA 2016 GLIA 2017;65:62–74     

Fyn kinase genetic ablation causes structural abnormalities in mature retina and defective Müller cell function

Fyn kinase is widely expressed in neuronal and glial cells of the brain, where it exerts multiple functional roles that affect fundamental physiological processes. The aim of our study was to investigate the, so far unknown, functional role of Fyn in the retina. We report that Fyn is expressed, in vivo, in a subpopulation of Müller glia. We used a mouse model of Fyn genetic ablation and Müller-enriched primary cultures to demonstrate that Fyn deficiency induces morphological alterations in the mature retina, a reduction in the thickness of the outer and inner nuclear layers and alterations in postnatal Müller cell physiology. These include shortening of Müller cell processes, a decrease in cell proliferation, inactivation of the Akt signal transduction pathway, a reduced number of focal adhesions points and decreased adhesion of these cells to the ECM. As abnormalities in Müller cell physiology have been previously associated to a compromised retinal function we evaluated behavioral responses to visual stimulation. Our results associate Fyn deficiency with impaired visual optokinetic responses under scotopic and photopic light conditions. Our study reveals novel roles for Fyn kinase in retinal morphology and Müller cell physiology and suggests that Fyn is required for optimal visual processing.     

High-affinity GABA uptake in retinal glial (Müller) cells of the guinea pig: electrophysiological characterization,immunohistochemical localization,and modeling of efficiency

Glial cells may act as important modulators of neuronal information processing, in particular, via fast uptake of neuronally released transmitters. Here, we characterize the electrogenic gamma-aminobutyric acid (GABA) transporters present in the plasma membranes of Müller (glial) cells of the guinea pig retina and present an estimate of their functional efficiency. The GABA-evoked whole-cell currents are voltage-dependent, with increasing amplitudes and decreasing affinity constants at more negative membrane potentials. The transmembranal GABA transport is concentration-dependent, with near-maximal currents at 100 microM GABA, and is dependent on extracellular sodium and chloride ions; the stoichiometry is 1 GABA/2 Na(+)/1 Cl(-). Immunohistochemical labeling and whole-cell voltage-clamp records reveal that Müller cells express both GAT-1 and GAT-3 (but not GAT-2), and that the transporter proteins are expressed predominantly at plasma membrane sites that, in situ, are localized in the outer retina where GABA uptake is performed exclusively by Müller cells. When extracellular GABA enters the cell interior, it evokes, via activation of the GABA transaminase, an NAD(P)H fluorescence signal selectively in the distal region of the Müller cells where their mitochondria are located. Using our experimental data, we simulated the GABA clearance from the extracellular space surrounding one Müller cell; these estimates show that a pulse of 100 microM extracellular GABA is fully cleared after 70 ms. It is suggested that Müller cells may be involved in the regulation of GABAergic transmission within the retina by providing a fast termination of GABAergic signaling via their highly efficient GABA uptake.     

Müller glial cells inhibit proliferation of retinal endothelial cells via TGF‐β2 and Smad signaling

Neovascularization is a sight‐threatening complication of ischemic proliferative retinopathies. Transforming growth factor (TGF)‐β, a cytokine with multiple functions in the retina, participates in the control of pathological angiogenesis and neovascularization. Retinal glial (Müller) cells produce TGF‐β2 under physiological and post‐ischemic conditions. To characterize glial cell‐derived mediators of angiogenesis regulation in glial‐endothelial interactions in the retina, we co‐cultured primary Müller cells and bovine microvascular retinal endothelial cells (BRECs). Müller cell‐derived TGF‐β2 was bound by the BRECs, which were found to express serine/threonine kinase TGF‐β receptors, and stimulated TGF‐β‐dependent anti‐proliferative signaling pathways. The proliferation of BRECs was attenuated by exogenous TGF‐β2 as well as by the presence of Müller cell culture media. The following intracellular signaling mechanisms were found to be involved in the anti‐angiogenic action of Müller cell‐derived TGF‐β2: (i) binding of TGF‐β2 to BRECs is mediated by the type‐II TGF‐β receptor, leading to (ii) activation and phosphorylation of receptor‐activated Smads; (iii) Müller cell‐derived TGF‐β2 activates Smad2 and Smad3 to (iv) attenuate the phosphorylation state of the MAP kinases, extracellular signal‐regulated kinase (ERK)‐1/‐2. Neutralizing TGF‐β or TGF‐β type‐II receptor or blocking the activation of Smads partially abrogated the effect of Müller cell‐conditioned media on BRECs. Together, our data suggest that Müller cells release TGF‐β2, inhibiting the proliferation of retinal endothelial cells via activation of Smad2/Smad3 and attenuation of ERK signaling. Given the context‐dependent action of TGF‐β2 on angiogenesis, our results may have implications for understanding the pathogenesis of retinal angiopathies, such as diabetic retinopathy, and the anti‐angiogenic role of TGF‐β therein. GLIA 2014;62:1476–1485     

microRNA expression in the neural retina: Focus on Müller glia

The neural retina hosts a unique specialized type of macroglial cell that not only preserves retinal homeostasis, function, and integrity but also may serve as a source of new neurons during regenerative processes: the Müller cell. Precise microRNA‐driven mechanisms of gene regulation impel and direct the processes of Müller glia lineage acquisition from retinal progenitors during development, the triggering of their response to retinal degeneration and, in some cases, Müller cell reprogramming and regenerative events. In this review we survey the recent reports describing, through functional assays, the regulatory role of microRNAs in Müller cell physiology, differentiation potential, and retinal pathology. We discuss also the evidence based on expression analysis that points out the relevance of a Müller glia–specific microRNA signature that would orchestrate these processes.     

Müller cell changes precede vascularization of the pigment epithelium in the dystrophic rat retina

In the Royal College of Surgeons rat with inherited retinal dystrophy, photoreceptor cell degeneration is accompanied by retinal pigment epithelial (RPE) cell alterations and Müller cell changes such as increased expression of glial fibrillary acidic protein (GFAP). Vascular changes such as vascularization of the RPE, vascular proliferation, and formation of vitreoretinal membranes (VRMs) are observed later. To study the relationship of Müller cell changes to the vascular alterations in the dystrophic retina, we used immunoperoxidase techniques and antibodies against GFAP and vimentin. Our study showed that during photoreceptor degeneration, Müller cells expressed small amounts of GFAP. As degeneration progressed, GFAP expression increased and morphological alterations occurred in Müller cells. Müller cell apical processes extended and proliferated in the subretinal space and contacted the apical surface of duplicated RPE cells. Later, GFAP reactive fibers surrounded retinal vessels apposed to the RPE. As the vessels became enmeshed within the RPE, the GFAP-positive perivascular processes disappeared. Eventually, the RPE-associated vessels became displaced into the inner retina where VRMs were sometimes observed. Immunoblots showed increased GFAP in dystrophic as compared with control retinas. Studies of vimentin distribution in the dystrophic retina showed results similar to the GFAP study. Moreover, the vimentin study suggested increased number of Müller cell processes in the dystrophic as compared with control retinas. The close temporal and anatomical relationships among Müller cell, RPE, and vascular changes in the dystrophic rat suggest a role for Müller cells in retinal neovascularization and proliferative retinopathy.     

VEGF release by retinal glia depends on both oxygen and glucose supply

Isolated retinae or isolated Müller cells were cultured in vitro, and vascular endothelial growth factor (VEGF) was assayed as protein (by ELISA) and as mRNA (by semi-quantitative RT-PCR). In both types of cultures, hypoxia (5% O2) resulted in an upregulated VEGF release. While the unstimulated VEGF secretion was virtually independent of glucose (0.125 - 25 mM), elevated glucose concentrations (10 - 25 mM) blocked most of the stimulatory effect of hypoxia on VEGF mRNA synthesis (determined in Müller cell cultures) as well as on VEGF release (in both retina and Müller cell cultures). It is concluded that in retinal glial (Müller) cells, being responsible for retinal VEGF synthesis (and, thus, for undesirable neovascularization), the metabolic effects of hypoxia can be compensated by a surplus of glucose.     

Tandem-pore domain potassium channels are functionally expressed in retinal (Müller) glial cells

Tandem-pore domain (2P-domain) K+-channels regulate neuronal excitability, but their function in glia, particularly, in retinal glial cells, is unclear. We have previously demonstrated the immunocytochemical localization of the 2P-domain K+ channels TASK-1 and TASK-2 in retinal Müller glial cells of amphibians. The purpose of the present study was to determine whether these channels were functional, by employing whole-cell recording from frog and mammalian (guinea pig, rat and mouse) Müller cells and confocal microscopy to monitor swelling in rat Müller cells. TASK-like immunolabel was localized in these cells. The currents mediated by 2P-domain channels were studied in isolation after blocking Kir, K(A), K(D), and BK channels. The remaining cell conductance was mostly outward and was depressed by acid pH, bupivacaine, methanandamide, quinine, and clofilium, and activated by alkaline pH in a manner consistent with that described for TASK channels. Arachidonic acid (an activator of TREK channels) had no effect on this conductance. Blockade of the conductance with bupivacaine depolarized the Müller cell membrane potential by about 50%. In slices of the rat retina, adenosine inhibited osmotic glial cell swelling via activation of A1 receptors and subsequent opening of 2P-domain K+ channels. The swelling was strongly increased by clofilium and quinine (inhibitors of 2P-domain K+ channels). These data suggest that 2P-domain K+ channels are involved in homeostasis of glial cell volume, in activity-dependent spatial K+ buffering and may play a role in maintenance of a hyperpolarized membrane potential especially in conditions where Kir channels are blocked or downregulated.     

BMP4 and CNTF are neuroprotective and suppress damage-induced proliferation of Müller glia in the retina

In response to acute damage, Müller glia in the chicken retina have been shown to be a source of proliferating progenitor-like cells. The secreted factors and signaling pathways that regulate this process remain unknown. The purpose of this study was to test whether secreted factors, which are known to promote glial differentiation during development, regulate the ability of Müller glia to proliferate and become retinal progenitors in response to acute damage in mature retina. We made intraocular injections of BMP4, BMP7, EGF, NGF, BDNF, or CNTF before or after a single, toxic dose of N-methyl-d-aspartate (NMDA) and assayed for proliferating progenitor-like cells within the retina. We found that injections of BMP4, BMP7, or CNTF, but not EGF, NGF, or BDNF, before NMDA treatment reduced the number of Müller glia that proliferated and gave rise to progenitor-like cells. CNTF and BMP4, but not NGF or BDNF, greatly reduced the number of cells destroyed by toxin treatment indicating that these factors protect retinal neurons from a severe excitotoxic insult. Injections of CNTF 5 days before NMDA treatment prevented neurotoxin-induced cell death and Müller glial proliferation, while injections of BMP4 had no protective effect. In addition, CNTF injected after NMDA treatment suppressed glial proliferation, while BMP4 did not. We conclude that BMP4 and CNTF, when applied before a toxic insult, act as neuroprotective agents and likely suppress the proliferative response of Müller glia to retinal damage by attenuating the retinal damage; protecting bipolar and amacrine neurons from NMDA-induced cell death. When applied after a toxic insult, CNTF suppressed glial proliferation independent of levels of retinal damage.