The Experts below are selected from a list of 135 Experts worldwide ranked by ideXlab platform
Joanne E Hogg - One of the best experts on this subject based on the ideXlab platform.
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effects of ischaemia on neurotransmitter release from the Isolated Retina
Journal of Neurochemistry, 2008Co-Authors: Michael J Neal, J R Cunningham, P H Hutson, Joanne E HoggAbstract:: The effects of "ischaemia" (glucose-free Krebs-bicarbonate medium gassed with N2/CO2) on the release of glutamate and other major neurotransmitters in the Retina were examined using the Isolated rat and rabbit Retina. Amino acid transmitters, acetylcholine, and dopamine were measured by HPLC. The release of glutamate, aspartate, GABA, and glycine from ischaemic Retinas was more than doubled after 30 min, and after 90 min of ischaemia the release of amino acids was approximately 15-20-fold that of control values. Ischaemia also produced large increases in the release of dopamine from both the rat and especially the rabbit Retina. In contrast, the release of acetylcholine from the rat Retina was significantly decreased by ischaemia, although the release of choline was increased. Because the ischaemia-induced release of glutamate, aspartate, and GABA from the rat Retina was completely Ca independent, and exposure of the Retina to high K (50 mM) did not stimulate amino acid release, it is concluded that the mechanisms underlying the ischaemia-induced release do not involve an initial release of K or an influx of calcium.
David R. Pepperberg - One of the best experts on this subject based on the ideXlab platform.
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Interphotoreceptor retinoid-binding protein (IRBP) promotes the release of all-trans retinol from the Isolated Retina following rhodopsin bleaching illumination
Experimental Eye Research, 2005Co-Authors: Nasser M. Qtaishat, Barbara Wiggert, David R. PepperbergAbstract:Abstract All- trans retinol generated in rod photoreceptors upon the bleaching of rhodopsin is known to move from the rods to the Retinal pigment epithelium (RPE), where it is enzymatically converted to 11- cis Retinal in the retinoid visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) contained in the extracellular compartment (interphotoreceptor matrix) that separates the Retina and RPE has been hypothesized to facilitate this movement of all- trans retinol, but the precise role of IRBP in this process remains unclear. To examine the activity of IRBP in the release of all- trans retinol from the rods, initially dark-adapted Isolated Retinas obtained from toad ( Bufo marinus ) eyes were bleached and then incubated in darkness for defined periods (5–180 min) in physiological saline (Ringer solution) supplemented with IRBP (here termed ‘IRBP I’) at defined concentrations (2–90 μ m ). Retinoids present in the Retina and extracellular medium were then determined by extraction and HPLC analysis. Preparations incubated with ≥10 μ m IRBP I showed a pronounced release of all- trans retinol with increasing period of incubation. As determined with 25 μ m IRBP I, the increase of all- trans retinol in the extracellular medium was accompanied by a significant decrease in the combined amount of all- trans Retinal and all- trans retinol contained in the Retina. This effect was not mimicked by unsupplemented Ringer solution or by Ringer solution containing 25 or 90 μ m bovine serum albumin. However, incubation with ‘IRBP II’, a previously described variant of IRBP with altered lectin-binding properties, led to the appearance of substantial all- trans retinol in the extracellular medium. The results suggest that in vivo, IRBP plays a direct role in the release of all- trans retinol from the rods during operation of the visual cycle.
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[61] Generation of rhodopsin and "artificial" visual pigments in electrophysiologically active photoreceptors
Methods in Enzymology, 2004Co-Authors: David R. PepperbergAbstract:Publisher Summary This chapter discusses the generation of rhodopsin and “artificial” visual pigments in electrophysiologically active Photoreceptors. The properties of these “artificial” visual pigments are of direct interest in understanding the chemistry of rhodopsin itself, in as much as they represent structurally perturbed forms of the native pigment. This chapter describes the methods used to analyze the physiological activities of exogenous chromophores in the Isolated Retina preparation: preparation of the Isolated Retina, light adaptation, and application of test compound and resulting electrophysiological effects. The sensitization appears to result directly from the regeneration event, through stimulation of a “photochemical” process of dark adaptation that normally operates in the intact eye. The Retina and its base of filter paper are positioned in a recording dish above a chlorided silver wire, which serves as a reference electrode. The preparation is maintained at a physiological temperature under a gentle stream of moistened oxygen; drying of the preparation is prevented by occasional additions of Ringer's solution to the Retina and surrounding filter paper.
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Retinol Kinetics in the Isolated Retina Determined by Retinoid Extraction and HPLC
Experimental Eye Research, 1997Co-Authors: Ting Ing L Okajima, David R. PepperbergAbstract:Abstract Suzuki et al. [Vis. Res.26, 425–9 (1986);Vis. Res.28, 1061–70 (1988)] have described a formaldehyde-based (HCHO-based) extraction procedure that efficiently recovers 11-cisRetinal initially present as rhodopsin chromophore in photoreceptor membranes. Using the Isolated Retina of the toad (Bufo marinus), we tested whether this procedure (‘HCHO’ method), in combination with a formaldehyde-free extraction procedure (‘i/h’ method) and the analysis of extracted retinoids by high performance liquid chromatography (HPLC), can account quantitatively for light-induced changes in retinoid levels and thus serve as an alternative to spectrophotometry for tracking the formation of all-transretinol in this intact rod preparation. Initially dark-adapted Retinas were incubated in bright light or in darkness and then analysed by homogenization and extraction using the HCHO and i/h methods. Combined data obtained using the two extraction procedures indicated a near-conservation of total retinoid recovered from dark-incubated and illuminated Retinas, and thus accounted for light-induced changes in retinoid levels. The HCHO procedure, employing formaldehyde, isopropanol and hexane, was similar to that described by Suzuki et al. and recovered Retinaldehydes including chromophoric 11-cisRetinal. The i/h procedure utilized isopropanol and hexane and, unlike the HCHO method, efficiently recovered all-transretinol. Illumination (onset at time zero) that produced an approximately exponential decline of 11-cisRetinal (time constant of 24 s) led to an increase and then a gradual decline in all-transRetinal. The normalized peak level of all-transRetinal, representing about 0.54 of the total molar quantity of recovered retinoid, developed with illumination periods of 10–80 s. The normalized level of all-transretinol reached ≃0.3 in Retinas illuminated for 1 min and, with longer illuminations (up to 30 min), exhibited an approximately exponential further growth to ≃0.9 with a time constant of 9.2 min. The results indicate the workability of the HCHO and i/h extraction procedures for tracking the in situ conversion of all-transRetinal to all-transretinol, a reaction thought to be important for both operation of the retinoid visual cycle and shut-off of the phototransduction cascade.
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Hydroxylamine-dependent inhibition of rhodopsin phosphorylation in the Isolated Retina.
Experimental Eye Research, 1992Co-Authors: David R. Pepperberg, Ting Ing L OkajimaAbstract:Abstract Hydroxylamine (NH2OH), a substance known to accelerate the decay of the metarhodopsin II bleaching intermediate of rhodopsin, was examined for its effect on the light-dependent phosphorylation of rhodopsin in the intact, Isolated Retina. Groups of ovine and bovine Retinas that had been pre-incubated in darkness with 32P-inorganic phosphate were supplemented with NH2OH at final concentrations of up to 20 m m , then irradiated and further incubated in darkness. Rod outer segments Isolated from the incubated Retinas were subjected to SDS-PAGE; the gel was analysed for 32P (autoradiography) and protein (Coomassie staining), to determine the specific radioactivity (ratio of 32P and protein levels; ‘32P/opsin’) of the opsin monomer band. Among Retinas of a given experimental group, 32P/opsin declined with increasing concentration of added NH2OH. The relative value of 32P/opsin exhibited by controls (0 m m NH2OH) was halved in the presence of about 1–2 m m NH2OH, and was reduced by ≥ 80% in the presence of 20 m m NH2OH. Supplementation of the Retina with 20 m m NH2OH 1 min after irradiation caused relatively little reduction in 32P/opsin. The results indicate that the light-dependent phosphorylation of rhodopsin in situ is substantially inhibited by NH2OH at millimolar levels. The data are discussed in relation to previous electrophysiological studies that have examined rod dark adaptation in NH2OH-treated Retinas.
John W Olney - One of the best experts on this subject based on the ideXlab platform.
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involvement of glutamate in ischemic neurodegeneration in Isolated Retina
Visual Neuroscience, 2003Co-Authors: Yukitoshi Izumi, John W Olney, Seth B Hammerman, Charity O Kirby, Ann Benz, Charles F ZorumskiAbstract:Retinal ischemia, a major cause of visual loss, is believed to result from overexcitation of glutamate receptors. However, under euglycemic and normoxic conditions, exogenously applied glutamate is not neurotoxic in the Retina. Under such conditions, exogenous glutamate typically causes glia swelling and requires very high concentrations to produce neurotoxicity. To determine whether ischemic conditions enhance the neurotoxicity of endogenous and exogenous glutamate, we examined the effects of simulated ischemia (deprivation of both glucose and oxygen) on Retinal morphology and lactate dehydrogenase (LDH) release. In an ex vivo rat Retinal preparation, glutamate was administered during simulated ischemia in the presence of riluzole, an inhibitor of glutamate release. Deprivation of both glucose and oxygen for 60 min at 30°C produced severe acute neurodegeneration. This neurodegeneration, characterized by bull's eye formation in the inner nuclear layer and spongy appearance in the inner plexiform layer, was prevented by the combination of MK-801 and DNQX, antagonists of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, indicating that the damage results from activation of both glutamate receptors. We also found that administration of glutamate pyruvate transaminase (alanine aminotransaminase) with pyruvate diminished the neurodegeneration during simulated ischemia. Furthermore, riluzole, an inhibitor of glutamate release, attenuated the neurodegeneration, suggesting the importance of endogenous glutamate in ischemic damage. In the presence of riluzole and simulated ischemia, exogenously applied glutamate failed to cause Muller cell swelling but was extremely neurotoxic. These results suggest that simulated ischemia enhances glutamate-mediated neurotoxicity in part by depressing glutamate uptake. When glutamate transport is impaired, sub-millimolar glutamate concentrations become profoundly neurotoxic.
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delayed excitotoxic neurodegeneration induced by excitatory amino acid agonists in Isolated Retina
Journal of Neurochemistry, 2002Co-Authors: Carmelo Romano, M T Price, John W OlneyAbstract:: Evidence from in vitro studies suggests that excitotoxic neuronal degeneration can occur by either an acute or delayed mechanism. Studies of the acute mechanism in Isolated chick embryo Retina using histological methods indicate that this process is rapidly triggered by activation of glutamate receptors of either the N-methyl-d-aspartate (NMDA) or non-NMDA subtypes. The delayed mechanism, studied primarily in cortical and hippocampal cell cultures prepared from embryonic rodent brain, requires activation of NMDA receptors. In these cell culture systems, stimulation of non-NMDA receptors does not rapidly trigger delayed neuronal degeneration, or does so only indirectly, via activation of NMDA receptors secondary to glutamate release. To provide a more valid basis for comparison of these two mechanisms, we have modified the Isolated chick embryo Retina model to permit studies of delayed as well as acute excitotoxic neurodegeneration. Retinas maintained for 24 h exhibited no morphological or biochemical signs of damage. Retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium at various times after exposure to agonists and normalized to total LDH in each Retina. Glutamate exposure (1 mM, 30 min) did not result in LDH release by the end of the exposure period, but LDH was released over the following 24 h. Briefer periods also led to substantial LDH release. Incubation in the presence of NMDA, or the non-NMDA agonists kainate (KA) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), led rapidly to delayed LDH release. NMDA and AMPA were more potent than glutamate, but high concentrations of glutamate led to more LDH release than high concentrations of these agonists. KA was a powerful excitotoxin, providing more LDH release than glutamate, NMDA, or AMPA at every concentration tested. The delayed LDH release induced by glutamate involved activation of both NMDA and non-NMDA receptors, as a combination of receptor-selective antagonists was necessary to provide complete blockade. These results indicate that glutamate, NMDA, AMPA, and KA all cause delayed as well as acute excitotoxic damage in the Retina. It is interesting that brief exposure to the non-NMDA receptor agonists, in relatively low concentrations, led to delayed LDH release. This is different than in other in vitro models of delayed excitotoxic neurodegeneration.
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the intact Isolated ex vivo Retina as a model system for the study of excitotoxicity
Progress in Retinal and Eye Research, 1998Co-Authors: Carmelo Romano, Quan Chen, John W OlneyAbstract:Abstract Excitotoxicity is defined as a mode of neural cell death triggered by overactivation of receptors for the amino acid transmitter glutamate. There is considerable evidence that excitotoxicity is responsible for cell death in several neuropathological states, including some Retinal diseases. The Isolated Retina, particularly from chick embryos, has been used extensively as an experimental system to characterize this process. This paper summarizes the use of Isolated Retina as a model system for studies of excitotoxicity from a theoretical and methodological point of view, and reviews results obtained from studies utilizing this system.
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ca2 independent excitotoxic neurodegeneration in Isolated Retina an intact neural net a role for cl and inhibitory transmitters
Molecular Pharmacology, 1998Co-Authors: Quan Chen, Peter D. Lukasiewicz, John W Olney, Todd Almli, Carmelo RomanoAbstract:Rapidly triggered excitotoxic cell death is widely thought to be due to excessive influx of extracellular Ca2+, primarily through the N -methyl-d-aspartate subtype of glutamate receptor. By devising conditions that permit the maintenance of Isolated Retina in the absence of Ca2+, it has become technically feasible to test the dependence of excitotoxic neurodegeneration in this intact neural system on extracellular Ca2+. Using biochemical, Ca2+ imaging, and electrophysiological techniques, we found that (1) rapidly triggered excitotoxic cell death in this system occurs independently of both extracellular Ca2+ and increases in intracellular Ca2+; (2) this cell death is highly dependent on extracellular Cl−; and (3) lethal Cl− entry occurs by multiple paths, but a significant fraction occurs through pathologically activated γ-aminobutyric acid and glycine receptors. These results emphasize the importance of Ca2+-independent mechanisms and the role that local transmitter circuitry plays in excitotoxic cell death.
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Ca2+-Independent Excitotoxic Neurodegeneration in Isolated Retina, an Intact Neural Net: A Role for Cl− and Inhibitory Transmitters
Molecular Pharmacology, 1998Co-Authors: Quan Chen, Peter D. Lukasiewicz, John W Olney, Todd Almli, Carmelo RomanoAbstract:Rapidly triggered excitotoxic cell death is widely thought to be due to excessive influx of extracellular Ca2+, primarily through the N -methyl-d-aspartate subtype of glutamate receptor. By devising conditions that permit the maintenance of Isolated Retina in the absence of Ca2+, it has become technically feasible to test the dependence of excitotoxic neurodegeneration in this intact neural system on extracellular Ca2+. Using biochemical, Ca2+ imaging, and electrophysiological techniques, we found that (1) rapidly triggered excitotoxic cell death in this system occurs independently of both extracellular Ca2+ and increases in intracellular Ca2+; (2) this cell death is highly dependent on extracellular Cl−; and (3) lethal Cl− entry occurs by multiple paths, but a significant fraction occurs through pathologically activated γ-aminobutyric acid and glycine receptors. These results emphasize the importance of Ca2+-independent mechanisms and the role that local transmitter circuitry plays in excitotoxic cell death.
Michael J Neal - One of the best experts on this subject based on the ideXlab platform.
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effects of ischaemia on neurotransmitter release from the Isolated Retina
Journal of Neurochemistry, 2008Co-Authors: Michael J Neal, J R Cunningham, P H Hutson, Joanne E HoggAbstract:: The effects of "ischaemia" (glucose-free Krebs-bicarbonate medium gassed with N2/CO2) on the release of glutamate and other major neurotransmitters in the Retina were examined using the Isolated rat and rabbit Retina. Amino acid transmitters, acetylcholine, and dopamine were measured by HPLC. The release of glutamate, aspartate, GABA, and glycine from ischaemic Retinas was more than doubled after 30 min, and after 90 min of ischaemia the release of amino acids was approximately 15-20-fold that of control values. Ischaemia also produced large increases in the release of dopamine from both the rat and especially the rabbit Retina. In contrast, the release of acetylcholine from the rat Retina was significantly decreased by ischaemia, although the release of choline was increased. Because the ischaemia-induced release of glutamate, aspartate, and GABA from the rat Retina was completely Ca independent, and exposure of the Retina to high K (50 mM) did not stimulate amino acid release, it is concluded that the mechanisms underlying the ischaemia-induced release do not involve an initial release of K or an influx of calcium.
Ari Koskelainen - One of the best experts on this subject based on the ideXlab platform.
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flash responses of mouse rod photoreceptors in the Isolated Retina and corneal electroretinogram comparison of gain and kinetics
Investigative Ophthalmology & Visual Science, 2012Co-Authors: Hanna Heikkinen, Frans Vinberg, Marja Pitkanen, Bertel Kommonen, Ari KoskelainenAbstract:PURPOSE: To examine the amplification and kinetics of murine rod photoresponses by recording ERG flash responses in vivo and ex vivo from the same Retina. We also aimed to evaluate the two available methods for isolating the rod signal from the ERG flash response, that is, pharmacology and paired flash method on the Isolated Retina. METHODS: Dark-adapted ERG responses to full-field flashes of green light were recorded from anesthetized (ketamine/xylazine) C57BL/6N mice. ERG flash responses to homogenous light stimuli arriving from the photoreceptor side were then recorded transRetinally from the same Retinas, Isolated and perfused with Ringer's or Ames' solution at 37°C. The responses were analyzed to determine the a-wave kinetics as well as the estimated flash sensitivity and kinetics of the full rod responses derived with the paired flash protocol. The analysis was complemented with pharmacologic blockade of glutamatergic transmission in the Isolated Retina. RESULTS: The a-waves were of comparable size, sensitivity and kinetics in vivo and in the Isolated Retina, but the onset of the b-wave was delayed in the Isolated Retina. The Lamb-Pugh activation constants determined for the a-waves were similar in both preparations. The kinetics of the derived photoreceptor responses were similar in both conditions, although the responses were consistently slightly slower ex vivo. This was not explicable as a direct effect of ketamine or xylazine on the photoreceptors or as their indirect effect through hyperglycemia, as tested on the Isolated Retina. CONCLUSIONS: Through comparison to the corneal ERG, the transRetinal ERG is a valuable tool for assaying the physiologic state of Isolated Retinal tissue. The rod photoreceptor responses of the intact Isolated Retina correspond well to those recorded in vivo. The origin of their faster kinetics compared to single cell recordings remains to be determined.
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mouse cone photoresponses obtained with electroretinogram from the Isolated Retina
Vision Research, 2008Co-Authors: Hanna Heikkinen, Soile Nymark, Ari KoskelainenAbstract:Abstract We characterize the dark-adapted photoresponses from mouse cones intact in the Isolated Retina, their virtually natural environment, by isolating pharmacologically the photoreceptor light responses from the electroretinogram (ERG). Due to the different photoresponse kinetics and sensitivity of rods and cones, the cone responses were readily attained by using a rod-saturating preflash. The stimulus wavelength (544 nm) was chosen to selectively stimulate the green sensitive (“M”-)pigment. Obtained responses were monophasic, showing fast kinetics (mean t p = 51 ms) and low sensitivity (fractional single-photon response ca. 0.23%). The amplification coefficient of cones (4.6 s −2 ) was very close to that of rods (5.6 s −2 ), while the dominant time constant of recovery was clearly smaller for cones (33 ms) than for rods (160 ms).
