2-Deoxy-D-glucose protects retinal ganglion cells against excitotoxicity

Caloric restriction mimicked by administration of 2-deoxy-D-glucose (2DG) has been shown to protect cerebral neurons against ischemia and excitotoxicity. This study examined the protective effects of pretreatment with 2DG on retinal neurons in N-methyl-D-aspartate (NMDA) excitotoxicity in rats. There was a significantly reduced number of TUNEL-labeled cells in the retinal ganglion cell layer 18 h after intravitreal injection of NMDA with 2DG pretreatment. At 7 days after NMDA, 2DG pretreatment significantly preserved neurons in the retinal ganglion cell layer and reduced immunoreactivity of glial fibrillary acidic proteins in retinas. Our findings demonstrate that caloric restriction mimicked by 2DG protects retinas from NMDA excitotoxicity.     

BDNF regulates NMDA receptor activity in developing retinal ganglion cells

Brain-derived neurotrophic factor (BDNF) modulates glutamate receptors of the NMDA type in many areas of the brain. We assessed whether BDNF exerts an effect on NMDA receptor properties in retinal ganglion cells during early postnatal development. Electrophysiological responses to the glutamate agonist NMDA (500 microM-2 mM) in retinal slices of wildtype and BDNF deficient mice (bdnf-/-) were recorded using the whole-cell patch-clamp technique. Retinal ganglion cells of bdnf-/- mice displayed significantly smaller NMDA currents than those of age-matched wildtype mice. Remarkably, NMDA receptor activity was restored by incubating retinal slices of bdnf-/- mice in BDNF (50 ng/ml) for 1-3 h. We suggest that BDNF plays a role in the activation of functional NMDA receptors in early ganglion cell development.     

The mechanism by which NBQX enhances NMDA currents in retinal ganglion cells

When the quinoxaline NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (F) quinoxaline), a KA/AMPA antagonist, is bath applied to the tiger salamander retina, a paradoxical action is evident in the light-evoked synaptic responses of ganglion cells: NBQX enhances excitatory synaptic currents at light onset observed under whole-cell voltage-clamp conditions in a perfused retinal slice preparation. This observation was surprising because synaptic inputs into ganglion cells that are mediated by KA/AMPA receptors are entirely blocked by NBQX. Thus, the NBQX-enhanced current is entirely mediated by NMDA receptors. The purpose of this study was to determine the mechanism(s) by which blocking KA/AMPA receptors appears to enhance NMDA currents. Using hyperosmotic sucrose stimulation to activate neurotransmitter release from the inner retina, we observed that NBQX augmented the sucrose-evoked response, suggesting that at least a component of this enhancement may reside in the inner retina. NBQX does not enhance NMDA currents activated by bath applied NMDA, demonstrating that the NBQX-induced enhancement does not result from modulation of NMDA receptors. Voltage-clamp studies, carried out at the appropriate holding potential, indicate that NBQX enhances glutamatergic transmission and reduces inhibitory inputs onto ganglion cells. In the presence of strychnine and picrotoxin, the NBQX-induced enhancement of NMDA currents is eliminated, suggesting that NBQX facilitates the expression of NMDA currents by a selective and partial reduction of inhibitory mechanisms. Additional studies suggest that part of the NMDA enhancement by NBQX is evident at the postsynaptic level, but a presynaptic component probably also participates, perhaps at the level of bipolar cell terminals. One way to account for this observation is to assume that a subpopulation of inhibitory amacrine cells requires KA/AMPA receptors exclusively for their synaptic activation: previous studies of sustained amacrine cells support this interpretation. Thus the NBQX-induced enhancement phenomenon may reflect a network-selective distribution of NMDA and KA/AMPA receptors among third-order neurons.     

Muller glia induce retinal progenitor cells to differentiate into retinal ganglion cells

Retinal progenitor cells could differentiate into various retinal cells that made cell-replacement therapy possible. Here, we investigated the role of cellular microenvironment on their regulation and differentiation and found that the percentage of proliferating cells and the percentage of retinal ganglion cells produced from them increased, when retinal progenitor cells were cocultured with Muller glia. Muller glia conditioned medium had the similar results. It is speculated that rather than traditional supportive roles, Muller glia may have an active regulatory role inducing retinal progenitor cells to proliferate and differentiate into ganglion cells by secreting some diffusible and membrane-associated factors. Identification of Muller glia-derived factors will be made to elucidate the molecular mechanisms of neurogenesis.     

Misplaced NMDA receptors in epileptogenesis contribute to excitotoxicity

Pharmacological blockade of NR2B-containing N-methyl-d-aspartate receptors (NMDARs) during epileptogenesis reduces neurodegeneration provoked in the rodent hippocampus by status epilepticus. The functional consequences of NMDAR activation are crucially influenced by their synaptic vs extrasynaptic localization, and both NMDAR function and localization are dependent on the presence of the NR2B subunit and its phosphorylation state. We investigated whether changes in NR2B subunit phosphorylation, and alterations in its neuronal membrane localization and cellular expression occur during epileptogenesis, and if these changes are involved in neuronal cell loss. We also explored NR2B subunit changes both in the acute phase of status epilepticus and in the chronic phase of spontaneous seizures which encompass the epileptogenesis phase. Levels of Tyr1472 phosphorylated NR2B subunit decreased in the post-synaptic membranes from rat hippocampus during epileptogenesis induced by electrical status epilepticus. This effect was concomitant with a reduced interaction between NR2B and post-synaptic density (PSD)-95 protein, and was associated with decreased CREB phosphorylation. This evidence suggests an extra-synaptic localization of NR2B subunit in epileptogenesis. Accordingly, electron microscopy showed increased NR2B both in extra-synaptic and pre-synaptic neuronal compartments, and a concomitant decrease of this subunit in PSD, thus indicating a shift in NR2B membrane localization. De novo expression of NR2B in activated astrocytes was also found in epileptogenesis indicating ectopic receptor expression in glia. The NR2B phosphorylation changes detected at completion of status epilepticus, and interictally in the chronic phase of spontaneous seizures, are predictive of receptor translocation from synaptic to extrasynaptic sites. Pharmacological blockade of NR2B-containing NMDARs by ifenprodil administration during epileptogenesis significantly reduced pyramidal cell loss in the hippocampus, showing that the observed post-translational and cellular changes of NR2B subunit contribute to excitotoxicity. Therefore, pharmacological targeting of misplaced NR2B-containing NMDARs, or prevention of these NMDAR changes, should be considered to block excitotoxicity which develops after various pro-epileptogenic brain injuries.     

Schwann cells exhibit excitotoxicity consistent with release of NMDA receptor agonists

Neurodegenerative effects of Schwann cells transplanted into the central nervous system have been observed previously. We report here that conditioned medium from Schwann cell cultures exhibit degenerative influences on hippocampal neurons. Aliquots of Schwann cell-conditioned medium compromised the morphologic integrity of the neurons, markedly elevated their intracellular calcium concentrations, and decreased their viability. The degenerative effects of Schwann cell medium on neuronal morphology and viability were blocked by N-methyl-D-aspartate (NMDA) receptor antagonists D-(-)-2-amino-5-phosphonopentanoic acid (D-APV) and 5,7-dicholorokynurenic acid (DCKA). Glutamate was detected in Schwann cell-conditioned medium at a concentration on the order of 10(-5) M. D-Amino acid oxidase (DAAOx) also attenuated the neurotoxicity exhibited by Schwann cells. These data suggest that Schwann cells release biologically relevant concentrations of excitotoxins that include glutamate and D-serine.     

Topography of pig retinal ganglion cells

In the present work we analyzed the distribution of retinal ganglion cells (RGCs) in the pig retina. RGCs were retrogradely labeled in vivo by injecting Fluoro-Gold into the optic nerve. RGC density and the distribution of RGCs in terms of soma size were analyzed. Different regions of the porcine retina were identified following analysis of the distribution of RGCs in terms of cell density and soma size: in the central retina, we found a high-density horizontal RGC band lying dorsal to the optic disc. Moreover, in this region, a high proportion of RCGs with small soma size was observed. From the central to the more peripheral retina, we observed a decrease in RGC density, together with a greater presence of RGCs with larger somas. The results of this study should prove to be useful as a foundation for future studies with the porcine retina as a model in ophthalmic research. The study also highlights the necessity to label the RGC population specifically with retrograde tracers in order to quantify precisely alterations in the cell population associated with experimental treatments.     

Correlated firing of retinal ganglion cells

Even in the absence of visual stimulation, retinal ganglion cells have a substantial maintained discharge. This maintained discharge is not generated independently within each ganglion cell, because the unstimulated activity of two neighboring ganglion cells can be remarkably correlated. These correlations show that two such cells respond together to strong, spontaneous signals from more distal retinal neurons and that, in some cases, ganglion cells even have effects on each other. Observations of correlated firing can give insights not only into the sources of maintained activity but also into retinal connections and signal processing. Correlating firing at the retinal level also has important implications for the use of correlation analysis to study connections between cells in higher visual centers. Much recent attention has focused on the role that correlated firing may play in forming appropriate, ordered connections to a target structure.     

Apolipoprotein E protects against NMDA excitotoxicity

Preclinical and clinical evidence implicates a role for endogenous apolipoprotein E in modifying the response of the brain to focal and global ischemia. To investigate whether apoE modulates the neuronal response to glutamate excitotoxicity, we exposed primary neuronal glial cultures and a neuronal cell line to biologically relevant concentrations of apolipoprotein E prior to NMDA exposure. In both of these paradigms, apolipoprotein E exerted partial protective effects. At neuroprotective concentrations, however, apolipoprotein E failed to block NMDA-induced calcium influx to the same magnitude as the NMDA receptor antagonist MK-801. These results suggest that one mechanism by which apolipoprotein E modifies the central nervous system response to ischemia may be by reducing glutamate-induced excitotoxicity.     

Intrinsically photosensitive retinal ganglion cells.

The recent discovery of melanopsin-expressing retinal ganglion cells that mediate the pupil light reflex has provided new insights into how the pupil responds to different properties of light. These ganglion cells are unique in their ability to transduce light into electrical energy. There are parallels between the electrophysiologic behavior of these cells in primates and the clinical pupil response to chromatic stimuli. Under photopic conditions, a red light stimulus produces a pupil constriction mediated predominantly by cone input via trans-synaptic activation of melanopsin-expressing retinal ganglion cells, whereas a blue light stimulus at high intensity produces a steady-state pupil constriction mediated primarily by direct intrinsic photoactivation of the melanopsin-expressing ganglion cells. Preliminary data in humans suggest that under photopic conditions, cones primarily drive the transient phase of the pupil light reflex, whereas intrinsic activation of the melanopsin-expressing ganglion cells contributes heavily to sustained pupil constriction. The use of chromatic light stimuli to elicit transient and sustained pupil light reflexes may become a clinical pupil test that allows differentiation between disorders affecting photoreceptors and those affecting retinal ganglion cells.     

Morphological identification and systematic classification of mammalian retinal ganglion cells. I. Rabbit retinal ganglion cells

Retinal ganglion cells (RGCs) convey visual signals to 50 regions of the brain. For reasons of interest and convenience, they constitute an excellent system for the study of brain structure and function. There is general agreement that, absent a complete “parts list,” understanding how the nervous system processes information will remain an elusive goal. Recent studies indicate that there are 30–50 types of ganglion cell in mouse retina, whereas only a few years ago it was still written that mice and the more visually oriented lagomorphs had less than 20 types of RGC. More than 30 years ago, I estimated that rabbits have about 40 types of RGC. The present study indicates that this number is much too low. I have employed the old but powerful method of Golgi-impregnation to rabbit retina, studying the range of component neurons in this already well-studied retinal system. Close quantitative and qualitative analyses of 1,142 RGCs in 26 retinas take into account cell body and dendritic field size, level(s) of dendritic stratification in the retina's inner plexiform layer, and details of dendritic branching. Ninety-one morphologies are recognized. Of these, at least 32 can be correlated with physiologically studied RGCs, dye-injected for morphological analysis. It is unlikely that rabbits have 91 types of RGC, but is argued here that this number lies between 60 and 70. The present study provides a “yardstick” for measuring the output of future molecular studies that may be more definitive in fixing the number of RGC types in rabbit retina.     

Central projections of cat retinal ganglion cells

The central projections of different groups of cat retinal ganglion cells were studied following small iontophoretic injections of horseradish peroxidase (HRP) into physiologically characterized sites. Analysis was restricted to labeled cells in the upper periphery of the nasal retina, contralateral to the injection site. Injections were made to the A lamina and C lamina of the dorsal lateral geniculate nucleus (LGNd-A,C), the geniculate wing (LGNd-W), the ventral lateral geniculate nucleus (LGNv), the pretectum (PT), and the superior colliculus (SC). The dendritic fields of alpha, beta, and epsilon cells were well labeled by the procedures we employed. A group, termed "g1," had somal sizes within the range of the smaller beta and epsilon cells, but dendritic morphologies distinct from either class. The g1 group may consist of a number of types, but our material provided no basis for further distinguishing them. Many cells were observed that had smaller somas; all had thin axons, and few had dendritic fields that labeled to any significant extent. We were not able to further distinguish these cells, and refer to this group, which may include a number of types, as "g2" cells. From the peripheral nasal retina, alpha cells project to LGNd-A, LGNd-C, PT, and SC. Beta cells project to LGNd-A, LGNd-C, and PT. Epsilon and g1 cells project to the LGNd-C, LGNd-W, LGNv, PT, and SC. We determined the total spatial density of cells in the region of the retina analyzed, using a Nissl-stained preparation. We then estimated the relative fraction of cells in each of the above groupings by injecting HRP throughout a cross section of the optic tract. Multiplying this relative fraction by the total spatial density gave an estimate of the spatial density of each of these groupings. From the spatial density of cells labeled from the injection site, we were able to estimate the fraction of cells of each retinal grouping that project to each of the zones investigated. By these calculations, almost all alpha cells from the upper nasal retina project to LGNd-A and LGNd-C; most project to SC, and about a third to PT. Beta cells, by contrast, project almost exclusively to LGNd-A, with about 10% going to LGNd-C, and about 1% to the PT. The great majority of epsilon cells, if not all, project to LGNd-W, and up to half of this population also project to the other zones noted above.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dissociation of retinal ganglion cells without enzymes

We describe here methods for dissociating retinal ganglion cells from adult goldfish and rat without proteolytic enzymes, and show responses of ganglion cells isolated this way to step-wise voltage changes and fluctuating current injections. Taking advantage of the laminar organization of vertebrate retinas, photoreceptors and other cells were lifted away from the distal side of freshly isolated goldfish retinas, after contact with pieces of membrane filter. Likewise, cells were sliced away from the distal side of freshly isolated rat retinas, after these adhered to a membrane filter. The remaining portions of retina were incubated in an enzyme-free, low Ca2+ solution, and triturated. After aliquots of the resulting cell suspension were plated, ganglion cells could be identified by dye retrogradely transported via the optic nerve. These cells showed no obvious morphological degeneration for several days of culture. Perforated-patch whole-cell recordings showed that the goldfish ganglion cells spike tonically in response to depolarizing constant current injections, that these spikes are temporally precise in response to fluctuating current injections, and that the largest voltage-gated Na+ currents of these cells were larger than those of ganglion cells isolated with a neutral protease.     

Early differentiation of retinal ganglion cells: an axonal protein expressed by premigratory and migrating retinal ganglion cells

A monoclonal antibody, RA4, was developed that recognizes retinal ganglion cell axons in the mature retina. Between embryonic days 3 and 9, the RA4 antigen was associated with cell bodies in certain regions of the retina in addition to the ganglion cell axons. The RA4-positive cells were of 3 types: an apolar cell adjacent to the ventricular surface, a bipolar cell that spanned the thickness of the retina, and a monopolar cell in the ganglion cell layer. Evidence suggests that these cells are premigratory and migrating retinal ganglion cells. The expression of the RA4 antigen is the earliest indicator of ganglion cell differentiation yet reported. The existence of RA4-positive apolar cells along the outer surface of the retina suggests that the ganglion cell phenotype is expressed as soon as the cell becomes postmitotic. Approximately 20% of the migrating ganglion cells were in pairs. The paired cells most likely arose from the terminal division of a germinal cell. One possibility suggested by these data is that a ganglion cell-specific germinal cell arises from a pluripotent germinal cell. Immunoblots and other analyses revealed the RA4 antigen to be a 140 kDa cytoplasmic protein in the retina. RA4 also recognized many long tract axons in the brain. In the brain, the RA4 epitope was observed on proteins with at least 7 different molecular weights. Evidence suggests that different cell types may express the RA4 antigen with slightly different molecular weights.     

Glutamate transporters and retinal excitotoxicity

Glutamate appears to play a major role in several degenerative retinal disorders. However, exogenous glutamate is only weakly toxic to the retina when glutamate transporters on Müller glial cells are operational. In an ex vivo rat retinal preparation, we previously found that exogenous glutamate causes Müller cell swelling but does not trigger excitotoxic neurodegeneration unless very high concentrations that overwhelm the capacity of glutamate transporters are administered. To determine the role of glutamate transporters in Müller cell swelling and glutamate-mediated retinal degeneration, we examined the effects of DL-threo-beta-benzyloxyaspartate (TBOA), an agent that blocks glutamate transport but that unlike most available transport inhibitors is neither a substrate for transport nor a glutamate receptor agonist. We found that TBOA triggered severe retinal neurodegeneration attenuated by ionotropic glutamate receptor antagonists. TBOA-induced neuronal damage was also diminished by riluzole, an agent that inhibits endogenous glutamate release. In the presence of riluzole, to inhibit glutamate release plus TBOA to block glutamate uptake, the addition of low concentrations of exogenous glutamate triggered severe excitotoxic neuronal damage without inducing Müller cell swelling. We conclude that TBOA-sensitive glutamate transporters play an important role in regulating the neurodegenerative effects of glutamate in the rat retina.     

Depth segregation of retinal ganglion cells projecting to mouse superior colliculus

The retinotectal projections in the mouse were analyzed with injections of horseradish peroxidase into the superior colliculus and of radioactive amino acids into the eye. At least 70% of the ganglion cells, and possibly all of them, were found to project to the superior colliculus, including ganglion cells of all sizes. Small injections revealed that ganglion cells of different sizes terminate at different levels in the superior colliculus. The small ganglion cells that form the vast majority of all cells project predominantly to the upper stratum griseum superficiale. A small population of mainly medium-sized and large ganglion cells project to the deep stratum griseum superficiale and to the stratum opticum. The ipsilateral projection is restricted to the deep stratum griseum superficiale and stratum opticum and consists predominantly of medium-sized and large ganglion cells.     

In vivo imaging of murine retinal ganglion cells

Current methods for in vivo retinal ganglion cells (RGCs) imaging involve either retrograde or intravitreal injection of chemical or biological tracers, which are invasive and may require repeated injection for serial long-term assessment. We have developed a confocal scanning laser ophthalmoscope technique (blue-light CSLO or bCSLO) to image retinal ganglion cells (RGCs) in mice expressing cyan fluorescent protein under the control of a Thy-1 promoter. Fluorescent spots corresponding to CFP-expressing retinal ganglion cells were discernable with the bCSLO. 96.1+/-2.6% of CFP expressing cells also were retrograde labeled with DiI indicating the bCSLO imaged fluorescent spots are RGCs. The imaging of Thy-1 promoter-driven CFP expression in these mice could serve as a sensitive indicator to reflect the integrity of RGCs, and provides a non-invasive method for longitudinal study of the mechanism of RGC degeneration and the effect of neuroprotective agents.