Pattern recognition of amino acid signatures in retinal neurons

Marc, RE; Murry, RF; Basinger, SF

doi:10.1523/jneurosci.15-07-05106.1995

Cited by 209 publications

(425 citation statements)

References 73 publications

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“…ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer. Scale bar=20 μm using these methods is proportional to the logarithm of the concentration of antigen in the tissue [34][35][36].…”

Section: Expression Of Glast and Eaat4mentioning

confidence: 99%

Glutamate uptake in retinal glial cells during diabetes

et al. 2005

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Aims: Glutamate recycling is a major function of retinal Müller cells. The aim of this study was to evaluate the expression and function of glutamate transporters during diabetes. Methods: Sprague-Dawley rats were rendered diabetic by a single dose of streptozotocin (50 mg/kg). Following 12 weeks of diabetes, immunolocalisation and mRNA expression of the two glial cell transporters, GLAST and EAAT4 were evaluated using indirect immunofluorescence and real-time PCR. The function of glutamate transport was investigated at 1, 4 and 12 weeks following induction of diabetes by measuring the level of uptake of the non-metabolisable glutamate analogue, D-aspartate, into Müller cells. Results: There was no difference in the localisation of either GLAST or EAAT4 during diabetes. Although there was a small apparent increase in expression of both GLAST and EAAT4 in diabetic retinae compared with controls this was not statistically significant. At 1, 4 and 12 weeks following diabetes, D-aspartate immunoreactivity was significantly increased in Müller cells of diabetic rats compared to controls (p<0.001). The EC 50 was found to increase by 0.304 log units in diabetic Müller cells compared with controls, suggesting that glutamate uptake is twice as efficient. Conclusions: These data suggest that there are alterations in glutamate transport during diabetes. However, these changes are unlikely to play a significant role in glutamate-induced neuronal excitoxicity during diabetes. These results suggest that although Müller cells undergo gliosis at an early stage of diabetes, one of the most important functions for maintaining normal retinal function is preserved within the retina.

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Section: Expression Of Glast and Eaat4mentioning

confidence: 99%

Glutamate uptake in retinal glial cells during diabetes

et al. 2005

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“…Serial thin 250-nm sections were fully etched in sodium methoxide and probed for D-aspartate, L-glutamate, Lglutamine, taurine, L-aspartate, glutathione using anti-hapten IgGs described in previous work (e.g., Marc et al, 1995;Marc and Cameron, 2002). Signals were visualized with calibrated silver-intensified 1-nm immunogold secondary IgGs, channels registered with Ͻ200-nm precision with PCI Geomatica (Richmond Hill, Canada) remote sensing code (for details, see Marc et al, 1995) and analyzed with in-house multivariate analysis code (CellKit V1.0.6) developed under IDL VM™ from RSI (Boulder, CO). Signal histograms were calibrated as intracellular concentration by remapping pixel values to known concentrations in ovalbumin-lysine-amino acid standard stacks similar to those fabricated in previous studies (Ottersen et al, 1992;Crook and Pow, 1997;Marc and Jones, 2002).…”

Section: Glutamate Uptake: D-aspartate Uptake Immunocytochemistrymentioning

confidence: 99%

“…Because glutamate is rapidly degraded to glutamine in Mü ller cells, we used D-aspartate as a substrate to determine glutamate uptake. Also, D-Asp was localized by immunocytochemistry using a specific antibody (Marc et al, 1995). Results of the uptake experiment are presented in Figure 4A,B.…”

Section: D-aspartate Uptake By Mü Ller Cellsmentioning

confidence: 99%

“…One advantage of the method (Marc and Jones, 2002;Jones et al, 2003) is that it allows for concurrent quantitative measures of multiple intracellular markers in the same cells. Calibrated small molecular immunocytochemistry can accurately detect concentrations in the range of 0.1 -10 mM (Marc et al, 1990(Marc et al, , 1995Marc and Jones, 2002). It is also possible to use pattern recognition methods to completely segment images into discrete cellular compartments (Marc et al, 1995), each of which can be individually sampled.…”

Section: D-aspartate Uptake By Mü Ller Cellsmentioning

confidence: 99%

“…Calibrated small molecular immunocytochemistry can accurately detect concentrations in the range of 0.1 -10 mM (Marc et al, 1990(Marc et al, , 1995Marc and Jones, 2002). It is also possible to use pattern recognition methods to completely segment images into discrete cellular compartments (Marc et al, 1995), each of which can be individually sampled. The same specimens illustrated in Figure 4A,B were segmented by pattern recognition and calibrated signal histograms acquired from 42 GLAST Ϫ/Ϫ Mü ller cells and 46 wildtype Mü ller cells and their processes (Fig.…”

Section: D-aspartate Uptake By Mü Ller Cellsmentioning

confidence: 99%

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Glutamate transport by retinal Müller cells in glutamate/aspartate transporter‐knockout mice

Sarthy

Pignataro

Pannicke

et al. 2004

Glia

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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 Mü ller cells in GLAST Ϫ/Ϫ mice, using biochemical, electrophysiological, and immunocytochemical techniques. Glutamate uptake assays indicated that the K m value for glutamate uptake was similar in wild-type and GLAST Ϫ/Ϫ mouse retinas, but the V max was ϳ50% lower in the mutant. In Na ϩ -free medium, the V max was further reduced by 40%. In patch-clamp recordings of dissociated Mü ller cells from GLAST Ϫ/Ϫ 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 Mü ller cells. [ 3 H]D-Aspartate uptake autoradiography, however, showed that Na ϩ -dependent, high-affinity transporters account for most of the glutamate uptake by Mü ller cells, and that Na ϩ -independent glutamate transport is negligible. Additional experiments showed that the residual glutamate uptake in Mü ller cells in the GLAST Ϫ/Ϫ 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 Mü ller cells, new Na ϩ -dependent, high-affinity glutamate transporters remain to be identified.

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Glutamate metabolic pathways in displaced ganglion cells of the chicken retina

Kalloniatis

Napper

1996

J. Comp. Neurol.

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Glutamate (E) is the putative amino acid neurotransmitter used by ganglion cells, photoreceptors, and bipolar cells. Aspartate (D) and glutamine (Q) are potential precursors of glutamate, and glutamate-utilizing neurons may use one or more of these amino acids to sustain production of glutamate. We used post-embedding immunocytochemistry for several amino acid neurotransmitters to characterize the amino acid signatures for displaced ganglion cells of the avian retina. We found two neurochemical signatures for displaced ganglion cells, EQ and EDQ, in mid-peripheral and far-peripheral retina, respectively. Differences in neurochemical signatures cannot be explained by the existence of two ganglion cell populations, and we propose that the two signature categories for the large-diameter displaced ganglion cells reflect variations in the aspartate precursor pool. The transamination reaction involved in glutamate production, aspartate/oxaloacetate and alpha-ketoglutarate/glutamate, requires an active TCA cycle, since the carbon skeleton of glutamate is derived from alpha-ketoglutarate, a TCA intermediary. We hypothesized that aspartate levels vary in the normal chicken retina because eccentricity-dependent differences in oxygen availability result in changes of alpha-ketoglutarate levels, and hence, alterations in the equilibrium of the transamination reaction. We tested this hypothesis by incubating isolated chicken retinas in anaerobic conditions and found elevated aspartate immunoreactivity in subpopulations of glutamate-utilizing neurons in the central retina. Under aerobic conditions, or in retinas placed directly into fixative, retinal samples from the central edge of the pecten did not show differential cellular staining for aspartate. We have, therefore, identified differences in neurochemical signatures for retinal neurons involving changes in active maintenance of precursor pools.

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Pattern recognition of amino acid signatures in retinal neurons

Cited by 209 publications

References 73 publications

Glutamate uptake in retinal glial cells during diabetes

Glutamate uptake in retinal glial cells during diabetes

Glutamate transport by retinal Müller cells in glutamate/aspartate transporter‐knockout mice

Glutamate metabolic pathways in displaced ganglion cells of the chicken retina

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