Light stimulation evokes two different calcium responses in Müller glial cells of the guinea pig retina

Intracellular calcium responses are a characteristic of glial activation upon neuronal activity. In acutely isolated preparations of the guinea pig retina, Müller glial cells displayed cytosolic calcium rises in response to repetitive light stimulation. The calcium rises consisted of two components, a slowly developing immediate response that occurred simultaneously over the whole length of all Müller cell fibers and a delayed fast response that originated in the ganglion cell layer and spread as a wave through the bodies of some Müller cells toward the outer processes in the photoreceptor layer. The slow calcium response was evoked by photoreceptor-to-glia signaling, resulting in a glutamate transporter- and zinc-mediated alteration in the membrane potential and an influx of calcium from the extracellular space. The fast calcium response was evoked by a release of calcium from intracellular stores, probably after activation of purinergic receptors. The data suggest that light stimulation of the retina causes glial activation by alterations in both the membrane potential and receptor-mediated mechanisms. The former may be implicated in glial support of the neuronal signal transfer from photoreceptors to ganglion cells (glial forward signaling), whereas the latter may constitute a glial feedback signaling from ganglion cells to photoreceptors.     

BMP‐ and TGFβ‐signaling regulate the formation of Müller glia‐derived progenitor cells in the avian retina

Müller glia‐derived progenitor cells (MGPCs) have the capability to regenerate neurons in the retinas of different vertebrate orders. The formation of MGPCs is regulated by a network of cell‐signaling pathways. The purpose of this study was to investigate how BMP/Smad1/5/8‐ and TGFβ/Smad2/3‐signaling are coordinated to influence the formation of MGPCs in the chick model system. We find that pSmad1/5/8 is selectively up‐regulated in the nuclei of Müller glia following treatment with BMP4, FGF2, or NMDA‐induced damage, and this up‐regulation is blocked by a dorsomorphin analogue DMH1. By comparison, Smad2/3 is found in the nuclei of Müller glia in untreated retinas, and becomes localized to the cytoplasm following NMDA‐ or FGF2‐treatment. These findings suggest a decrease in TGFβ‐ and increase in BMP‐signaling when MGPCs are known to form. In both NMDA‐damaged and FGF2‐treated retinas, inhibition of BMP‐signaling suppressed the proliferation of MGPCs, whereas inhibition of TGFβ‐signaling stimulated the proliferation of MGPCs. Consistent with these findings, TGFβ2 suppressed the formation of MGPCs in NMDA‐damaged retinas. Our findings indicate that BMP/TGFβ/Smad‐signaling is recruited into the network of signaling pathways that controls the formation of proliferating MGPCs. We conclude that signaling through BMP4/Smad1/5/8 promotes the formation of MGPCs, whereas signaling through TGFβ/Smad2/3 suppresses the formation of MGPCs.     

Müller cells express the cannabinoid CB2 receptor in the vervet monkey retina

The presence of the cannabinoid receptor type 1 (CB1R) has been largely documented in the rodent and primate retinae in recent years. There is, however, some controversy concerning the presence of the CB2 receptor (CB2R) within the central nervous system. Only recently, CB2R has been found in the rodent retina, but its presence in the primate retina has not yet been demonstrated. The aim of this study was twofold: 1) to characterize the distribution patterns of CB2R in the monkey retina and compare this distribution with that previously reported for CB1R and 2) to resolve the controversy on the presence of CB2R in the neural component of the retina. We therefore thoroughly examined the cellular localization of CB2R in the vervet monkey (Chlorocebus sabeus) retina, using confocal microscopy. Our results demonstrate that CB2R, like CB1R, is present throughout the retinal layers, but with striking dissimilarities. Double labeling of CB2R and glutamine synthetase shows that CB2R is restricted to Müller cell processes, extending from the internal limiting membrane, with very low staining, to the external limiting membrane, with heavy labeling. We conclude that CB2R is indeed present in the retina but exclusively in the retinal glia, whereas CB1R is expressed only in the neuroretina. These results extend our knowledge on the expression and distribution of cannabinoid receptors in the monkey retina, although further experiments are still needed to clarify their role in retinal functions. J. Comp. Neurol. J. Comp. Neurol. 521:2399–2415, 2013. © 2013 Wiley Periodicals, Inc.     

The adenylate cyclase inhibitor MDL-12330A has a non-specific effect on glycine transport in Müller cells from the retina

Müller glial cells express two transport systems for glycine (Gly): one with low affinity and another identified as GLYT1 with high affinity. The latter colocalizes with NMDA receptors in the CNS. Gly is considered as an obligatory coagonist at NMDA receptors, and, hence, the Gly transport system could contribute to the modulation of glutamate (Glu) excitatory transmission in the vertical pathways of the retina. For this reason, the regulation of Gly transport by cAMP was studied. We report here a non-specific effect of MDL-12330A, a compound reported to inhibit adenylate cyclase (AC), on Gly transport in Müller glia. This effect might be due to a toxic action on the cells, decreasing cell viability, and not to a specific inhibition of the adenylate cyclase. Non-specific effects of this drug should be considered when the participation of cAMP in any biological process is studied. We have clearly demonstrated that cAMP does not participate in the regulation of Gly transport in Müller glia.     

Müller glial cell reactivation in Xenopus models of retinal degeneration

A striking aspect of tissue regeneration is its uneven distribution among different animal classes, both in terms of modalities and efficiency. The retina does not escape the rule, exhibiting extraordinary self‐repair properties in anamniote species but extremely limited ones in mammals. Among cellular sources prone to contribute to retinal regeneration are Müller glial cells, which in teleosts have been known for a decade to re‐acquire a stem/progenitor state and regenerate retinal neurons following injury. As their regenerative potential was hitherto unexplored in amphibians, we tackled this issue using two Xenopus retinal injury paradigms we implemented: a mechanical needle poke injury and a transgenic model allowing for conditional photoreceptor cell ablation. These models revealed that Müller cells are indeed able to proliferate and replace lost cells following damage/degeneration in the retina. Interestingly, the extent of cell cycle re‐entry appears dependent on the age of the animal, with a refractory period in early tadpole stages. Our findings pave the way for future studies aimed at identifying the molecular cues that either sustain or constrain the recruitment of Müller glia, an issue of utmost importance to set up therapeutic strategies for eye regenerative medicine.     

Prospective purification and characterization of Müller glia in the mouse retina regeneration assay

Reactive gliosis is an umbrella term for various glia functions in neurodegenerative diseases and upon injury. Specifically, Müller glia (MG) in some species readily regenerate retinal neurons to restore vision loss after insult, whereas mammalian MG respond by reactive gliosis—a heterogeneous response which frequently includes cell hypertrophy and proliferation. Limited regeneration has been stimulated in mammals, with a higher propensity in young MG, and in vitro compared to in vivo, but the underlying processes are unknown. To facilitate studies on the mechanisms regulating and limiting glia functions, we developed a strategy to purify glia and their progeny by fluorescence‐activated cell sorting. Dual‐transgenic nuclear reporter mice, which label neurons and glia with red and green fluorescent proteins, respectively, have enabled MG enrichment up to 93% purity. We applied this approach to MG in a mouse retina regeneration ex vivo assay. Combined cell size and cell cycle analysis indicates that most MG hypertrophy and a subpopulation proliferates which, over time, become even larger in cell size than the ones that do not proliferate. MG undergo timed differential genomic changes in genes controlling stemness and neurogenic competence; and glial markers are downregulated. Genes that are potentially required for, or associated with, regeneration and reactive gliosis are differentially regulated by retina explant culture time, epidermal growth factor stimulation, and animal age. Thus, MG enrichment facilitates cellular and molecular studies which, in combination with the mouse retina regeneration assay, provide an experimental approach for deciphering mechanisms that possibly regulate reactive gliosis and limit regeneration in mammals.     

Glial fibrillary acidic (GFA) protein in Müller glia. Immunofluorescence study of the goldfish retina

Glial fibrillary acidic protein, the subunit of intermediate filaments specific for astrocytes, was localized by immunofluorescence in the Müller glia of goldfish retina. Based on previous studies reporting the localization in Müller glia of carbonic anhydrase C, an oligodendrocyte marker, we suggest that the main type of neuroglia in the retina combines properties which in the brain are specific for astrocytes and oligodendrocytes.     

Neuron-specific distribution of P2X7 purinergic receptors in the monkey retina

Extracellular ATP is a signaling molecule, working through P2X purinoceptors in the nervous system. P2X7 is a major subtype of the purinoceptors in the brain, where it is expressed mostly in glia cells and considered to work as a trigger of cytolysis. In the rodent retina, however, P2X7 is expressed in several classes of neurons including ganglion cells. In the present study we identified cells immunopositive for P2X7 by double immunolabeling. Immunoreactivity for P2X7 was observed in the inner nuclear layer (INL), the inner plexiform layer (IPL), and the ganglion cell layer (GCL). In the INL, strongly immunopositive cells corresponded to the subpopulation of horizontal cells. In the IPL, fine processes were immunopositive. In the GCL, most of the ganglion cells showed P2X7 immunoreactivity. At the ultrastructural level, immunoreactivity was confirmed in the cytoplasm of ganglion cells. No P2X7 immunoreactivity was found in non-neural cells, i.e., Müller cells or microglia. The immunohistochemical distribution of other purinoceptor subtypes (P2X1, P2X2, and P2X4) was also examined in the monkey retina. Immunoreactivity for P2X1 was strongly detected in a band, in sublamina a of the IPL. The band existed at almost the same level as tyrosine hydroxylase immunoreactivity, but did not seem to actually overlap. P2X2 was not expressed in the retina, and P2X4 was only faintly expressed at the scleral margin of the INL. Because P2X7 in the primate retina is expressed exclusively in neurons, it may in this location be involved in neural transmission rather than in cytolysis, as found for glia cells.     

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.     

Sodium-dependent glutamate transport in Müller glial cells: regulation by phorbol esters

The regulation of the Na⁺-dependent high affinity glutamate/aspartate transporter system expressed in cultured Müller glia cells from chick retina was studied. Treatment of the cells with the Ca²⁺/diacylglycerol dependent protein kinase C (PKC) activator, phorbol 12-tetradecanoil-13-acetate (TPA) produced a decrease in [³H]-aspartate uptake which was reversed by staurosporine and partially by H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochoride], two PKC inhibitors. Long-term treatment with TPA resulted in a drastic decrease in the uptake activity, correlated with a substantial fall in the expression of the transporter protein. These findings suggest that PKC is involved in transport modulation at two different levels: phosphorylation and transporter expression in retinal Müller glial cells.     

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     

Müller glia as an important source of cytokines and inflammatory factors present in the gliotic retina during proliferative vitreoretinopathy

Retinal gliosis is characterized by biochemical and physiological changes that often lead to Müller glia proliferation and hypertrophy and is a feature of many neuro‐degenerative and inflammatory diseases such as proliferative vitreoretinopathy (PVR). Although Müller glia are known to release inflammatory factors and cytokines, it is not clear whether cytokine production by these cells mirrors the pattern of factors present in the gliotic retina. Lysates from normal cadaveric retina and gliotic retinal specimens from patients undergoing retinectomy for treatment of PVR, the Müller cell line MIO‐M1 and four human Müller glial cell preparations isolated from normal retina were examined for their expression of cytokines and inflammatory factors using semi‐quantitative dot blot antibody arrays and quantitative arrays. Comparative analysis of the expression of inflammatory factors showed that in comparison with normal retina, gliotic retina exhibited greater than twofold increase in 24/102 factors examined by semiquantitative arrays, and a significant increase in 19 out of 27 factors assessed by quantitative methods (P < 0.05 to P < 0.001). It was observed that with the exception of some chemotactic factors, the majority of cytokines and inflammatory factors were produced by Müller glia in vitro and included G‐CSF, MCP‐1, PDGF‐bb, RANTES, VEGF, and TGFβ2. These results showed that a large number of inflammatory factors expressed by Müller glia in vitro are upregulated in the gliotic retina, suggesting that targeting the production of inflammatory factors by Müller glia may constitute a valid approach to prevent neural damage during retinal gliosis and this merits further investigations. GLIA 2016;64:495–506     

Activation of P2Y receptors stimulates potassium and cation currents in acutely isolated human Müller (glial) cells.

The ability of various neurotransmitters/neuroactive substances to induce fast, transient rises of Ca(2+)-activated K(+) currents (I(BK)) caused by release of Ca(2+) from intracellular stores was investigated in Müller glial cells of the human retina. Müller cells were enzymatically isolated from retinas of healthy donors or of patients with proliferative vitreoretinopathy, and the transmembrane ionic currents were recorded using the whole-cell and the cell-attached patch-clamp techniques. The results of the screening experiments indicate that human Müller cells express, in addition to GABA(A) and perhaps glutamatergic and cholinergic receptors, predominantly P2 receptors. ATP and other nucleotides exerted two effects on membrane currents: repetitive transient increases of the I(BK) amplitude and, in a subpopulation of cells investigated, the appearance of a transient cation conductance at negative potentials. ATP and UTP increased dose-dependently the I(BK) amplitude with half-maximal effects at 0.33 and 0.50 microM, respectively. Since several different P2 receptor agonists increased the I(BK), it is assumed that human Müller cells express a mixture of different types of P2Y receptors. In cell-attached patches, extracellular application of ATP or UTP transiently increased the open probability of single putative BK channels. The increase of I(BK) and the appearance of the cation conductance in whole-cell records were abolished when intracellular Ca(2+) was buffered by a high-EGTA pipette solution or when IP(3) was included in the pipette solution. The expression of agonist-evoked transient cation currents was found to be stronger in cells from patients as compared to cells from healthy donors. It is concluded that human Müller glial cells express P2Y receptors that, via IP(3) formation, cause intracellular Ca(2+) release. The increased intracellular Ca(2+) concentration stimulates the activity of BK channels and may induce opening of cation channels. Both the ATP-induced activity of BK channels and the increased expression of Ca(2+)-gated cation channels may be important in respect to proliferative Müller cell gliosis.     

Axon-glia communication evokes calcium signaling in olfactory ensheathing cells of the developing olfactory bulb

Olfactory ensheathing cells (OECs) accompany receptor axons in the olfactory nerve and promote axonal growth into the central nervous system. The mechanisms underlying the communication between axons and OECs, however, have not been studied in detail yet. We investigated the effect of activity-dependent neuronal transmitter release on Ca(2+) signaling of OECs in acute mouse olfactory bulb slices using confocal Ca(2+) imaging. TTX-sensitive axonal activity upon electrical nerve stimulation triggers a rise in cytosolic Ca(2+) in OECs, which can be mimicked by application of DHPG, an agonist of metabotropic glutamate receptors (mGluRs). Both stimulation- and DHPG-induced Ca(2+) transients in OECs were abolished by depletion of intracellular Ca(2+) stores with cyclopiazonic acid (CPA). The mGluR(1)-specific antagonist CPCCOEt completely inhibited DHPG-evoked Ca(2+) transients, but reduced stimulation-induced Ca(2+) transients only partly, suggesting the involvement of another neurotransmitter. Application of ATP evoked CPA-sensitive Ca(2+) transients in OECs, which were inhibited by the P2Y(1)-specific antagonist MRS2179. Co-application of CPCCOEt and MRS2179 almost completely blocked the stimulation-induced Ca(2+) transients, indicating that they were mediated by mGluR(1) and P2Y(1) receptors. Our results show that OECs are able to respond to olfactory nerve activity with an increase in cytosolic Ca(2+) due to glutamate and ATP release.