Differential Effects of HCN Channel Block on On and Off Pathways in the Retina as a Potential Cause for Medication-Induced Phosphene Perception.

Purpose: Phosphene perception is a characteristic side effect of heart rate-reducing medication that acts on hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels. It is hypothesized that these phosphenes are caused by blocking HCN channels in photoreceptors and neurons of the retina, yet the underlying changes in visual signal processing in the retina caused by the HCN channel block are still unknown. Methods: We examined the effects of pharmacologic HCN channel block on the encoding of visual signals in retinal ganglion cells by recording ganglion cell spiking activity from isolated mouse retinas mounted on multielectrode arrays. Spontaneous activity and responses to various visual stimuli were measured before, during, and after administration of 3 µM ivabradine. Results: Retinal ganglion cells generally showed slower response kinetics and reduced sensitivity to high temporal frequencies under ivabradine. Moreover, ivabradine differentially affected the sensitivity of On and Off ganglion cells. On cells showed reduced response gain, whereas Off cells experienced an increase in response threshold. In line with these differential effects, Off cells, in contrast to On cells, also showed reduced baseline activity during visual stimulation and reduced spontaneous activity. Furthermore, Off cells, but not On cells, showed increased burst-like spiking activity in the presence of ivabradine. Conclusions: Our data suggest that pharmacologic HCN channel block in the retina leads to a shift in the relative activity of the On and Off pathways of the retina. We hypothesize that this imbalance may underlie the medication-induced perception of phosphenes.

Sensitivity to image recurrence across eye-movement-like image transitions through local serial inhibition in the retina.

Standard models of stimulus encoding in the retina postulate that image presentations activate neurons according to the increase of preferred contrast inside the receptive field. During natural vision, however, images do not arrive in isolation, but follow each other rapidly, separated by sudden gaze shifts. We here report that, contrary to standard models, specific ganglion cells in mouse retina are suppressed after a rapid image transition by changes in visual patterns across the transition, but respond with a distinct spike burst when the same pattern reappears. This sensitivity to image recurrence depends on opposing effects of glycinergic and GABAergic inhibition and can be explained by a circuit of local serial inhibition. Rapid image transitions thus trigger a mode of operation that differs from the processing of simpler stimuli and allows the retina to tag particular image parts or to detect transition types that lead to recurring stimulus patterns.

Delayed-rectifier K channels contribute to contrast adaptation in mammalian retinal ganglion cells.

Retinal ganglion cells adapt by reducing their sensitivity during periods of high contrast. Contrast adaptation in the firing response depends on both presynaptic and intrinsic mechanisms. Here, we investigated intrinsic mechanisms for contrast adaptation in OFF Alpha ganglion cells in the in vitro guinea pig retina. Using either visual stimulation or current injection, we show that brief depolarization evoked spiking and suppressed firing during subsequent depolarization. The suppression could be explained by Na channel inactivation, as shown in salamander cells. However, brief hyperpolarization in the physiological range (5-10 mV) also suppressed firing during subsequent depolarization. This suppression was selectively sensitive to blockers of delayed-rectifier K channels (K(DR)). In somatic membrane patches, we observed tetraethylammonium-sensitive K(DR) currents that activated near -25 mV. Recovery from inactivation occurred at potentials hyperpolarized to V(rest). Brief periods of hyperpolarization apparently remove K(DR) inactivation and thereby increase the channel pool available to suppress excitability during subsequent depolarization.

Spectral and temporal sensitivity of cone-mediated responses in mouse retinal ganglion cells.

The retina uses two photoreceptor types to encode the wide range of light intensities in the natural environment. Rods mediate vision in dim light, whereas cones mediate vision in bright light. Mouse photoreceptors include only 3% cones, and the majority of these coexpress two opsins (short- and middle-wavelength sensitive, S and M), with peak sensitivity to either ultraviolet (360 nm) or green light (508 nm). The M/S-opsin ratio varies across the retina but has not been characterized functionally, preventing quantitative study of cone-mediated vision. Furthermore, physiological and behavioral measurements suggested that mouse retina supports relatively slow temporal processing (peak sensitivity, ∼ 2-5 Hz) compared to primates; however, past studies used visible wavelengths that are inefficient at stimulating mouse S-opsin. Here, we measured the M/S-opsin expression ratio across the mouse retina, as reflected by ganglion cell responses in vitro, and probed cone-mediated ganglion cell temporal properties using ultraviolet light stimulation and linear systems analysis. From recordings in mice lacking rod function (Gnat1(-/-), Rho(-/-)), we estimate ∼ 70% M-opsin expression in far dorsal retina, dropping to <5% M-opsin expression throughout ventral retina. In mice lacking cone function (Gnat2(cpfl3)), light-adapted rod-mediated responses peaked at ∼ 5-7 Hz. In wild-type mice, cone-mediated responses peaked at ∼ 10 Hz, with substantial responsiveness up to ∼ 30 Hz. Therefore, despite the small percentage of cones, cone-mediated responses in mouse ganglion cells are fast and robust, similar to those in primates. These measurements enable quantitative analysis of cone-mediated responses at all levels of the visual system.

NMDA receptor contributions to visual contrast coding.

In the retina, it is not well understood how visual processing depends on AMPA- and NMDA-type glutamate receptors. Here we investigated how these receptors contribute to contrast coding in identified guinea pig ganglion cell types in vitro. NMDA-mediated responses were negligible in ON alpha cells but substantial in OFF alpha and delta cells. OFF delta cell NMDA receptors were composed of GluN2B subunits. Using a novel deconvolution method, we determined the individual contributions of AMPA, NMDA, and inhibitory currents to light responses of each cell type. OFF alpha and delta cells used NMDA receptors for encoding either the full contrast range (alpha), including near-threshold responses, or only a high range (delta). However, contrast sensitivity depended substantially on NMDA receptors only in OFF alpha cells. NMDA receptors contribute to visual contrast coding in a cell-type-specific manner. Certain cell types generate excitatory responses using primarily AMPA receptors or disinhibition.

Signalling of sphingosine-1-phosphate in Müller glial cells via the S1P/EDG-family of G-protein-coupled receptors.

Signalling of sphingosine-1-phosphate (S1P) via G-protein-coupled receptors of the Endothelial Differentiation Gene family differentially regulates cellular processes such as migration, proliferation and morphogenesis in a variety of cell types. Proliferation and migration of retinal Müller glial cells are involved in pathological events such as proliferative vitreoretinopathy and proliferative diabetic retinopathy. Investigation of possible functional roles of S1P receptors might thus open new insights into Müller cell pathophysiology. Here we show that cultured Müller cells from the guinea pig retina respond to application of S1P with an increase in the intracellular calcium content in a concentration-dependent manner (EC(50) 11nM). This calcium increase consists of two components; an initial fast peak and a slow plateau component. The initial transient is caused by a release of calcium from intracellular stores and is suppressed by U-73122, a selective phospholipase C inhibitor. The slow plateau component is caused by a calcium influx. These results suggest that the S1P-induced calcium response in Müller cells partially involves signalling via G-protein-coupled receptors. Moreover, S1P slightly induced Müller cell migration but no proliferation. Thus, the data indicate that Müller cells might be involved in S1P signalling in the retina.

Transmission of phase-coupling accuracy from the auditory nerve to spherical bushy cells in the Mongolian gerbil.

The phase of low-frequency sinusoids is encoded in phase-coupled discharges of spherical bushy cells (SBCs) of the anteroventral cochlear nucleus and transmitted to the medial superior olive, where binaural input-coincidence is used for processing of sound source localization. SBCs are innervated by auditory nerve fibers through large, excitatory synapses (endbulbs of Held) and by inhibitory inputs, which effectively reduce SBC discharge rates. Here we monitor presynaptic potentials of endbulb-terminals and postsynaptic spikes of SBCs in extracellular single unit recordings in vivo. We compare postsynaptic phase-coupling of SBCs and their presynaptic immediate auditory nerve input. In all but one SBC discharge rates at the characteristic frequency were reduced pre-to-postsynaptically and phase-coupling accuracy was increased in one-third of them. We investigated the contribution of systemic inhibition on spike timing in SBCs by iontophoretic application of glycine- and GABA-receptor antagonists (strychnine, bicuculline). Discharge rate increased in one-third of the units during antagonist application, which was accompanied by a deterioration of phase-coupling accuracy in half of those units. These results suggest that the phase-coupling accuracy is improved in a subpopulation of SBCs during transmission from the auditory nerve to the SBCs by reduction of spike rates.

Calcium signaling of thyrocytes is modulated by TSH through calcium binding protein expression.

TSH is an important stimulus to maintain thyroid epithelial differentiation. Impairment of TSH signal transduction can cause thyroid pathologies such as hot nodules, goiter and hyperthyroidism. In a gene expression study in Fischer rat thyroid cells (FRTL-5) using cDNA microarrays we found a TSH-dependent regulation of several calcium binding proteins, S100A4, S100A6 and annexin A6. Expression of these genes in FRTL-5 and regulation by TSH was confirmed with LightCycler qPCR and Western blotting. The differential expression of S100A4 was confirmed for cultured primary human thyrocytes. Calcium-imaging experiments showed that prestimulation with TSH attenuates ATP-elicited P2Y-mediated calcium signaling. Experiments with thapsigargin, TSH and calcium-free perfusion excluded an involvement of other purinergic receptors or an involvement of SERCA regulation. Instead, we find a correlation between S100A4 expression and the effects of TSH on calcium signaling. Overexpression of S100A4 in FRTL-5 and shRNA-mediated knockdown of S100A4 in follicular thyroid cancer cells (FTC133) confirm the ability of S100A4 to attenuate calcium signals. Under repeated stimulations with ATP the calcium retention of these cells is also modulated by S100A4, suggesting a role of S100A4 as calcium buffering protein. As a biological consequence of S100A4 overexpression we detected reduced ATP-stimulated cFos induction. Taken together, the results suggest that S100A4 and other calcium binding proteins are part of a signaling network connecting TSH signaling to calcium-mediated events which play a role in thyroid physiology like H2O2 production or even thyroid cancer.

Neurite branch retraction is caused by a threshold-dependent mechanical impact.

Recent results indicate that, in addition to chemical cues, mechanical stimuli may also impact neuronal growth. For instance, unlike most other cell types, neurons prefer soft substrates. However, the mechanisms responsible for the neuronal affinity for soft substrates have not yet been identified. In this study, we show that, in vitro, neurons continuously probe their mechanical environment. Growth cones visibly deform substrates with a compliance commensurate with their own. To understand the sensing of stiff substrates by growth cones, we investigated their precise temporal response to well-defined mechanical stress. When the applied stress exceeded a threshold of 274 +/- 41 pN/microm(2), neurons retracted and re-extended their processes, thereby enabling exploration of alternative directions. A calcium influx through stretch-activated ion channels and the detachment of adhesion sites were prerequisites for this retraction. Our data illustrate how growing neurons may detect and avoid stiff substrates--as a mechanism involved in axonal branch pruning--and provide what we believe is novel support of the idea that mechanics may act as guidance cue for neuronal growth.

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.

Expression of CXCL8, CXCR1, and CXCR2 in neurons and glial cells of the human and rabbit retina.

PURPOSE: Several eye diseases are accompanied by inflammatory processes. The authors examined the expression of the proinflammatory chemokine CXCL8 and the corresponding receptors in healthy human retinas, in cellular membranes from patients with proliferative vitreoretinopathy (PVR) or human glial cell cultures and in an animal model of PVR in rabbit eyes. METHODS: The authors used immunohistochemical methods, Western blotting, RT-PCR, and real time RT-PCR to characterize the expression of CXCL8, CXCR1, and CXCR2 in human and rabbit retinas. Functionality of the receptors in cultured glial cells was tested by Ca(2+) imaging. RESULTS: Immunohistochemical examinations of normal human and rabbit retinas revealed a distinct expression of CXCR1 and CXCR2 in several neuronal cell types. CXCL8 mRNA was demonstrated only by RT-PCR in normal retinas, and receptor expression was confirmed by Western blotting and RT-PCR. The presence of CXCR1 and CXCR2, but not CXCL8, was detected by immunostaining in glial fibrillary acidic protein-positive glial cells of cellular PVR membranes. Immunoreactivity for CXCL8, CXCR1, and CXCR2 was observed in virtually all cultured glial cells and in the human Müller cell line MIO-M1. Müller cells responded to the application of CXCL8 with increased cytosolic Ca(2+) concentrations. In PVR rabbit retinas, CXCR1 expression is increased in Müller cells, and CXCL8 and CXCR2 are strongly expressed in microglial cells. CONCLUSIONS: Expression of CXCL8 and CXCL8 receptors in glial cells of human PVR membranes and rabbit PVR retinas suggests an involvement in glial reactivity. Furthermore, the prominent expression of CXCR1 and CXCR2 in neurons of the healthy human and rabbit retina suggests additional physiological functions.

Pathological effects of glyoxalase I inhibition in SH-SY5Y neuroblastoma cells.

In Alzheimer's disease (AD), in aging, and under conditions of oxidative stress, the levels of reactive carbonyl compounds continuously increase. Accumulating carbonyl levels might be caused by an impaired enzymatic detoxification system. The major dicarbonyl detoxifying system is the glyoxalase system, which removes methylglyoxal in order to minimize cellular impairment. Although a reduced activity of glyoxalase I was evident in aging brains, it is not known how raising the intracellular methylglyoxal level influences neuronal function and the phosphorylation pattern of tau protein, which is known to be abnormally hyperphosphorylated in AD. To simulate a reduced glyoxalase I activity, we applied an inhibitor of glyoxalase I, p-bromobenzylglutathione cyclopentyl diester (pBrBzGSCp(2)), to SH-SY5Y neuroblastoma cells to induce chronically elevated methylglyoxal concentrations. We have shown that 10 microM pBrBzGSCp(2) leads to a fourfold elevation of the methylglyoxal level after 24 hr. In addition, glyoxalase I inhibition leads to reduced cell viability, strongly retracted neuritis, increase in [Ca(2+)](i), and activation of caspase-3. However, pBrBzGSCp(2) did not lead to tau "hyper"-phosphorylation despite activation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase but rather activated protein phosphatases 2 and induced tau dephosphorylation at the Ser(202)/Thr(205) and Ser(396)/Ser(404) epitopes. Preincubation with the carbonyl scavenger aminoguanidine prevented tau dephosphorylation, indicating the specific effect of methylglyoxal. Also, pretreatment with the inhibitor okadaic acid prevented tau dephosphorylation, indicating that methylglyoxal activates PP-2A. In summary, our data suggest that a reduced glyoxalase I activity mimics some changes associated with neurodegeneration, such as neurite retraction and apoptotic cell death.

Glial cell-mediated spread of retinal degeneration during detachment: a hypothesis based upon studies in rabbits.

In human subjects with peripheral retinal detachments, visual deficits are not restricted to the detached retina but are also present in the non-detached tissue. Based upon studies on a rabbit model of rhegmatogenous retinal detachment, we propose a glial cell-mediated mechanism of spread of retinal degeneration into non-detached retinal areas which may also have importance for the understanding of alterations in the human retina. Both detached and attached portions of the rabbit retina display photoreceptor cell degeneration and cystic degeneration of the innermost layers. An inverse mode of photoreceptor cell degeneration in the attached tissue suggests a disturbed support of the photoreceptor cells by Müller cells which show various indications of gliosis (increased expression of intermediate filaments, cell hypertrophy, decreased plasma membrane K(+) conductance, increased Ca(2+) responsiveness to purinergic stimulation) in both detached and attached tissues. We propose that gliotic alterations of Müller cells contribute to the degeneration of the attached retina, via disturbance of glial homeostasis mechanisms. A down-regulation of the K(+) conductance of Müller cells may prevent effective retinal K(+) and water clearance, and may favor photoreceptor cell degeneration and edema development.

The activation of IL-8 receptors in cultured guinea pig Müller glial cells is modified by signals from retinal pigment epithelium.

Interleukin 8 (IL-8, CXCL8) is a pro-inflammatory chemokine which attracts neutrophils to sites of inflammation via an activation of the G-protein-coupled receptors, CXCR1 and CXCR2. However, both IL-8 and IL-8 receptors are widely expressed in various tissues and cell types, and have been suggested to be involved in other functions such as angiogenesis, tumor growth, or brain pathology. We examined the expression of IL-8 and IL-8 receptors in highly enriched primary cultures of guinea pig Muller glial cells. Immunoreactivity for CXCL8, CXCR1 and CXCR2 was observed in all cultured Muller cells. The expression of CXCL8 was confirmed by PCR, and the secretion of the CXCL8 protein from Muller cells was revealed by ELISA. Western blots showed prominent bands at approximately 40 kDa by using antibodies specific for human CXCR1 and CXCR2, and the expression of a putative CXCR2 receptor in Muller cells was confirmed by PCR. Furthermore, cultured Muller cells responded to application of recombinant human IL-8 with an increase of the cytosolic Ca(2+) concentration. If supernatants of cultured human retinal pigment epithelium (RPE) cells were applied to the Muller cell cultures, no obvious changes were observed in the CXCL8, CXCR1 and CXCR2 expression but (i) Muller cell proliferation was stimulated, and (ii) there was an increased number of CXCL8-responsive Muller cells and the amplitudes of the evoked calcium responses were enhanced. It is concluded that Muller glial cells may participate in the inflammatory response(s) of the retina during ocular diseases, and that this contribution may be modified by interactions with RPE cells.

Physiological properties of retinal Muller glial cells from the cynomolgus monkey, Macaca fascicularis--a comparison to human Muller cells.

Retinae from rabbits and laboratory rodents are often used as 'models' of the human retina, although there are anatomical differences. To test whether monkey eyes provide a better model, a physiological study of Muller glial cells was performed comparing isolated cells and retinal wholemounts from the cynomolgus monkey, Macaca fascicularis and from man. The membrane conductance of Muller cells from both species was dominated by inward and outward K(+) currents. Cells displayed glutamate uptake currents and responded to nucleotides by intracellular Ca(2+) increases. However, there were also species differences, such as a lack of GABA(A) receptors and of Ca(2+)-dependent K(+) currents in monkey cells. Thus, the use of Muller cells from cynomolgus monkeys may be advantageous for investigating a few specific properties; in general, monkey cells are no more similar to human cells than those from standard laboratory animals.

Resensitization of P2Y receptors by growth factor-mediated activation of the phosphatidylinositol-3 kinase in retinal glial cells.

PURPOSE: To determine whether activation of receptor tyrosine kinases enhances the responsiveness of purinergic P2Y receptors in Muller glial cells, known to induce Muller cell proliferation. METHODS: The P2Y receptors of primary cultured Muller cells of the guinea pig were desensitized by short (30 seconds to 10 minutes)- and long (24 or 48 hours)-term application of adenosine 5'-triphosphate (ATP). Recordings of ATP-evoked intracellular calcium responses showed whether short-term application of different growth factors evoke a resensitization of the receptors. Coapplication of pharmacologic inhibitors showed whether activation of protein kinases is involved in receptor resensitization. RESULTS: Both short- and long-term incubation with ATP induced a significant P2Y receptor desensitization, which was indicated by a strongly reduced intracellular calcium mobilization and which lasted for at least 48 hours. However, the receptors were significantly resensitized after short-term application of platelet-derived, epidermal, or nerve growth factors. The growth factor-mediated resensitization was dependent on an intact cytoskeleton and on the activation of protein phosphatases and of the phosphatidylinositol-3 kinase (PI3K), but was independent of the activation of protein kinase C, src kinases, or extracellular signal-regulated kinases. CONCLUSIONS: The results show that activation of receptor tyrosine kinases causes, via activation of PI3K and protein phosphatases, a resensitization of P2Y receptors formerly desensitized by agonist application. The growth factor-mediated resensitization may underlie the previously observed enhanced responsiveness of P2Y receptors in retinal glial cells in experimental retinal detachment and proliferative vitreoretinopathy and may contribute to the induction of reactive gliosis and Muller cell proliferation.

Glutamate transport by retinal Muller cells in glutamate/aspartate transporter-knockout mice.

Glutamate transporters are involved in maintaining extracellular glutamate at a low level to ensure a high signal-to-noise ratio for glutamatergic neurotransmission and to protect neurons from excitotoxic damage. The mammalian retina is known to express the excitatory amino acid transporters, EAAT1-5; however, their specific role in glutamate homeostasis is poorly understood. To examine the role of the glial glutamate/aspartate transporter (GLAST) in the retina, we have studied glutamate transport by Muller cells in GLAST-/- mice, using biochemical, electrophysiological, and immunocytochemical techniques. Glutamate uptake assays indicated that the Km value for glutamate uptake was similar in wild-type and GLAST-/- mouse retinas, but the Vmax was approximately 50% lower in the mutant. In Na+-free medium, the Vmax was further reduced by 40%. In patch-clamp recordings of dissociated Muller cells from GLAST-/- mice, application of 0.1 mM glutamate evoked no current showing that the cells lacked functional electrogenic glutamate transporters. The result also indicated that there was no compensatory upregulation of EAATs in Muller cells. [3H]D-Aspartate uptake autoradiography, however, showed that Na+-dependent, high-affinity transporters account for most of the glutamate uptake by Muller cells, and that Na+-independent glutamate transport is negligible. Additional experiments showed that the residual glutamate uptake in Muller cells in the GLAST-/- mouse retina is not due to known glutamate transporters-cystine-glutamate exchanger, ASCT-1, AGT-1, or other heteroexchangers. The present study shows that while several known glutamate transporters are expressed by mammalian Muller cells, new Na+-dependent, high-affinity glutamate transporters remain to be identified.

Glutamate-evoked alterations of glial and neuronal cell morphology in the guinea pig retina.

Neuronal activity is accompanied by transmembranous ion fluxes that cause cell volume changes. In whole mounts of the guinea pig retina, application of glutamate resulted in fast swelling of neuronal cell bodies in the ganglion cell layer (GCL) and the inner nuclear layer (INL) (by approximately 40%) and a concomitant decrease of the thickness of glial cell processes in the inner plexiform layer (IPL) (by approximately 40%) that was accompanied by an elongation of the glial cells, by a thickening of the whole retinal tissue, and by a shrinkage of the extracellular space (by approximately 18%). The half-maximal effect of glutamate was observed at approximately 250 mum, after approximately 4 min. The swelling was caused predominantly by AMPA-kainate receptor-mediated influx of Na+ into retinal neurons. Similar but transient morphological alterations were induced by high K+ and dopamine, which caused release of endogenous glutamate and subsequent activation of AMPA-kainate receptors. Apparently, retinal glutamatergic transmission is accompanied by neuronal cell swelling that causes compensatory morphological alterations of glial cells. The effect of dopamine was elicitable only during light adaptation but not in the dark, and glutamate and high K+ induced strong ereffects in the dark than in the light. This suggests that not only the endogenous release of dopamine but also the responsiveness of glutamatergic neurons to dopamine is regulated by light-dark adaptation. Similar morphological alterations (neuronal swelling and decreased glial process thickness) were observed in whole mounts isolated immediately after experimental retinal ischemia, suggesting an involvement of AMPA-kainate receptor activation in putative neurotoxic cell swelling in the postischemic retina.

Neuropeptide Y-evoked proliferation of retinal glial (Muller) cells.

BACKGROUND: Glial cells in human retinas and in fibrocellular membranes from patients with proliferative vitreoretinopathy (PVR) have been described to upregulate their expression of Y1 receptors for neuropeptide Y (NPY) (Soler et al.: Glia 39:320, 2002). However, it is unknown whether Y1 receptor activation causes proliferation of retinal glial cells. We investigated whether NPY exerts a proliferation-stimulating effect on retinal glial cells, and compared the NPY-evoked signaling with the signaling of purinergic P2Y receptors. METHODS: Proliferation assays using bromodeoxyuridine were carried out on primarily cultured Muller glial cells of the guinea pig, in the absence and presence of blockers of Y1 receptors, of receptor tyrosine kinases (RTKs), of mitogen-activated protein kinases (MAPKs) and of phosphatidylinositol-3 kinase (PI3K). RESULTS: NPY exerted a biphasic effect on Muller cell proliferation. At low concentrations (0.1 ng/ml and 1 ng/ml) it decreased the proliferation rate of the cells, while at higher concentration (100 ng/ml) it increased Muller cell proliferation. The NPY-evoked proliferation was mediated by Y1 receptor stimulation and by activation of the p44/p42 MAPKs and partially of the p38 MAPK. Moreover, Y1 receptor-induced activation of PI3K as well as transactivations of the platelet-derived and the epidermal growth factor RTKs were necessary for full mitogenic effect of NPY. Y1 and P2Y receptors share partially common signal transduction pathways in Muller cells. CONCLUSION: It is suggested that NPY may be involved in stimulation of retinal glial cell proliferation during PVR when it is released at higher amounts into the injured retina.

Early glial cell reactivity in experimental retinal detachment: effect of suramin.

PURPOSE: In a rabbit model of retinal detachment, early Müller glial cell reactivity was monitored-specifically, changes in membrane features-to determine whether these changes involve an upregulation of purinergic P2 receptor-mediated responses and whether all or some of these alterations could be blocked by suramin or pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS). In addition, the immune cell reactivity (microglial cells and blood-derived immune cells) was monitored. METHODS: A local retinal detachment was induced by subretinal injection of a sodium hyaluronate solution. Three, 24, 48, and 72 hours after surgery, Müller cells were acutely isolated, and patch-clamp records of the whole-cell potassium currents were made. The presence of P2 receptor-mediated responses was determined by measuring extracellular adenosine triphosphate (ATP)-induced membrane current increases, and by recording of ATP-induced calcium responses at the vitreal surface of retinal wholemounts. The density of isolectin B(4)-labeled immune cells was determined in the nerve fiber layer of retinal wholemounts. RESULTS: Within 24 hours of detachment, Müller cell reactivity was evident. The cells downregulated the density of their inwardly rectifying potassium currents to 60% and 47% of the control value at 48 hours and 72 hours of detachment, respectively. This downregulation was accompanied by an enhanced incidence of cells which showed calcium and current responses after ATP application (control: 14%; 24 hours of detachment: 42%; 72 hours of detachment: 80%). Müller cell hypertrophy was apparent at 48 and 72 hours of detachment. Application of suramin during surgery inhibited the downregulation of potassium currents, but not the elevated responsiveness to extracellular ATP; PPADS had no effect. Suramin also inhibited the inflammatory response that was induced by the surgical procedure and that was apparent by the increased number of immune cells. CONCLUSIONS: Reactive responses of Müller cells occur within 24 hours of detachment. Suramin inhibits several (but not all) reactive glial alterations and therefore may represent one candidate for further investigations in the search for drugs that limit detrimental effects of immune cell activation and Müller cell gliosis during retinal detachment.