Rapid glutamatergic alterations in the neural retina induced by retinal detachment

PURPOSE: Retinal detachment induces neurochemical changes in the neural retina over a span of days to weeks. However, little information is available on the acute response in the retina to detachment. METHODS: Distribution of the neurotransmitters glutamate, glycine, and gamma-aminobutyric acid (GABA) and the metabolic amino acids aspartate and glutamine was examined immunocytochemically from 5 to 30 minutes and at 3 hours after retinal detachment in a salamander eyecup preparation. RESULTS: Glutamate showed a rapid depletion from neuronal cell bodies in detached retina, whereas Müller cells, which normally sequester and metabolize glutamate, showed increased immunolabeling for glutamine. Changes occurred exclusively in detached retinal regions of the eyecup. Aspartate, a precursor for glutamate synthesis, also showed decreased labeling in neuronal cell bodies in detached retinal regions, although these changes were not as striking as those observed for glutamate. In contrast, the distributions of the inhibitory amino acid neurotransmitters glycine and GABA were not affected appreciably by acute retinal detachment. CONCLUSIONS: These results indicate that retinal detachment induces rapid, localized alterations in the glutamatergic system of the neural retina that are consistent with a massive efflux of neuronal glutamate and concomitant alterations in glutamate metabolism. An acute efflux of neuronal glutamate in detached retina could contribute to excitotoxicity and to the initiation of structural alterations and changes in gene expression; it is also consistent with reported neurochemical changes associated with longer term retinal detachment.     

Quantification of amino acid neurochemistry secondary to NMDA or betaxolol application

Background: Alterations in retinal amino acid neurochemistry are an indicator of metabolic function. Glutamate is the primary excitatory amino acid neurotransmitter within the retina, and excessive levels of glutamate can potentially cause excitotoxicity, in particular, through the N‐methyl‐D‐aspartate (NMDA) subtype of glutamate receptor. Anomalies in NMDA receptor function have been implicated as causing many neurodegenerative disorders, and overactivation leads to neuronal death secondary to metabolic insult. Several pharmaceutical agents have been proposed as potential neuroprotective agents against excitotoxicity (e.g. betaxolol), yet any effects such drugs have on retinal neurochemistry have not been determined. Therefore, the aim of this study was to quantify the changes in retinal amino acid neurochemistry secondary to the application of NMDA with and without betaxolol. Methods: Functional NMDA channel activation was confirmed in both amacrine and ganglion cells by quantifying the entry into these neurones of a channel permeable probe (agmatine: 1‐amino‐4‐guanidobutane [AGB]). By probing serial thin sections with immunoglobulins targeting AGB, glutamate, γ‐aminobutyric acid (GABA) and glycine, it was possible to simultaneously study the neurochemical characteristic as well as the NMDA‐evoked AGB responses of different neurochemical populations of inner retinal neurones. Results: The authors have previously shown no accumulation of glutamate or GABA within Müller cells following NMDA application. Herein they report altered GABA and glycine immunoreactivity, but not glutamate immunoreactivity within neurones of the amacrine and ganglion cell layers following NMDA application. Finally, the addition of betaxolol did not significantly alter the normal neurochemistry of the retina. Conclusion: The retina possesses intrinsic mechanisms that allow it to maintain metabolic integrity during short periods of high NMDA application.     

Transmitter-gated currents of GABAergic amacrine-like cells in chick retinal cultures.

A subpopulation of cells developing in dissociated neuronal cultures prepared from 8-day-old embryonic chick retinae can be identified as putative in vitro counterparts of GABAergic amacrine cells by immunocytochemical and autoradiographic markers and by their electrophysiological responses to transmitter agonists. In the present study, transmitter-gated conductances expressed by these neurons were examined using the whole-cell patch-clamp technique. At negative holding potentials, the excitatory amino acid agonists N-methyl-D-aspartate (NMDA), kainate quisqualate, and glutamate induced inward currents with reversal potentials close to 0 mV in most of the cells selected for recording. NMDA-evoked responses were selectively blocked by the noncompetitive inhibitor MK 801 and by Mg2+ (in a voltage-dependent manner) and were potentiated in the presence of submicromolar concentrations of glycine. Glutamate apparently interacted with both NMDA and non-NMDA type receptors. All cells tested responded to the inhibitory transmitters GABA and glycine. Both inhibitory agonists could be shown to activate chloride conductances. Responses to GABA and glycine were specifically inhibited in the presence of bicuculline and strychnine, respectively. Thus, GABAergic neurons in retinal cultures express at least two different excitatory amino acid receptors--NMDA and non-NMDA--and two different inhibitory amino acid receptors--the GABAA and the glycine receptor. The results demonstrate the ability of the cultured neurons to develop an apparently mature phenotype and contribute to the understanding of the functional properties of GABAergic amacrine cells in the vertebrate retina.     

Neuronal migration and glial remodeling in degenerating retinas of aged rats and in nonneovascular AMD

PURPOSE: To demonstrate structural and immunocytochemical changes associated with light-induced degeneration in albino rat retinas and human AMD retinas. METHODS: Retinas from Wistar rats aged 3, 6, or 10 months were examined by immunocytochemistry, with antibodies to neuronal and glial markers. Results were compared with human nonneovascular AMD retinas. RESULTS: In aging rat retinas, many photoreceptors were lost in response to normal ambient light exposure. Photoreceptor loss was preceded by loss of RPE cells. Müller cells extended processes through gaps in Bruch's membrane, into the choroid. immunolabeling for gamma-aminobutyric acid (GABA), the glycine transporter Glyt-1, and the rod bipolar cell marker PKC revealed the presence of numerous neuronal somata and processes that appeared to have migrated into the choroidal region. Processes of presumptive ganglion cells remodeled and stratified in the choroid, where strong labeling for synaptic vesicle antigens was present. Myelination of retinal ganglion cell axons was also observed, especially in the peripheral retina. In AMD retinas, glial rearrangement and displacement of neurons suggestive of their migration were also observed. CONCLUSIONS: In response to loss of RPE and photoreceptor cells, adult retinal neurons migrate out of the retina along remodeled processes of Müller cells. The presence of synaptic vesicle antigens suggests the formation of new synapses between migrating neurons. The myelination is probably due to the ingress of Schwann cells from the sclera. The presence of some similar changes in human AMD retinas suggests that these findings are of broad significance for determining the likely events in transplantation of neurons in the human retina and elsewhere.     

Amino acid neurochemistry of the vertebrate retina

The dominant neurochemicals involved in encoding sensory information are the amino acid neurotransmitters, glutamate, γ-aminobutyrate (GABA) and glycine which mediate fast point-to-point synaptic transmission in the retina and other parts of the central nervous system. The relative abundance of these neurochemicals and the existence of neuronal and glial uptake mechanisms as well as a plethora of receptors support the key role these neurochemicals play in shaping neural information. However, in addition to subserving neurotransmitter roles, amino acids subserve normal metabolic/cellular functions, may be precursors for other amino acids, and may also be associated with protein synthesis. Post-embedding immunocytochemistry of small molecules has allowed the characterization of multiple amino acid profiles within subpopulations of neurons in the vertebrate retina. The general theme emerging from these studies is that the retinal through pathway uses glutamate as its neurotransmitter, and the lateral elements, GABA and/or glycine. Co-localization studies using quantitative immunocytochemistry have shown that virtually all neuronal space can be accounted for by the three dominant amino acids. In addition, co-localization studies have demonstrated that there are no purely aspartate, glutamine, alanine, leucine or ornithine immunoreactive neurons and thus these amino acids are likely to act as metabolites and may sustain glutamate production through a multitude of enzymatic pathways. The mapping of multiple cellular metabolic profiles during development or in degenerating retinas has shown that amino acid neurochemistry is a sensitive marker for metabolic activity. In the degenerating retina, (RCS retina), neurochemical anomalies were evident early in development (from birth), even before photoreceptors mature at PND6-8 implying a generalized metabolic dysfunction. Identification of metabolic anomalies within subpopulation of neurons is now possible and can be used to investigate a multitude of retinal functions including amino acid metabolic and neurochemical changes secondary to external insult as well as to expand our understanding of the intricate interrelationship between neurons and glia.     

Neurochemical heterogeneity of retinal bipolar cells.

BACKGROUND: Retinal bipolar cells show neurochemical changes in response to retinal insult, which could have profound effects on retinal function or recovery after insult however, the neurochemistry of bipolar cells is incompletely understood. METHODS The distribution of the amino acid neurotransmitters glutamate, gamma-aminobutyric acid (GABA), and glycine, and the metabolic amino acids, aspartate and glutamine, was examined immunocytochemically in the retina of the tiger salamander, a major model for the study of retinal anatomy and function. RESULTS: Each amino acid showed a unique distribution among retinal bipolar cells. All bipolar cells appear to contain glutamate and, under appropriate conditions, may also contain aspartate. GABA and glycine were restricted to a subset of conventionally placed bipolar cells located in the inner nuclear layer, but were not present in displaced bipolar cells located in the outer nuclear layer. In contrast, high levels of glutamine were almost exclusively restricted to displaced bipolar cells. Analysis of co-localization patterns revealed several different amino acid signatures within the bipolar cell population. CONCLUSIONS: The striking neurochemical heterogeneity among bipolar cells suggests important differences in signaling and metabolism that could affect cell survival, plasticity, and functional recovery from retinal insult in a cell-type-specific manner.     

Neurochemical changes following postmortem ischemia in the rat retina

Glutamate and gamma-aminobutyric acid (GABA) are the dominant amino acids in the retina and brain. The manufacturing and degradation pathways of both of these amino acids are intricately linked with the tricarboxylic acid cycle leading to rapid redistribution of these amino acids after metabolic insult. Postmortem ischemia in mammalian retina predominantly results in a loss of glutamate and GABA from neurons and accumulation of these amino acids within Müller cells. This accumulation of glutamate and GABA in Müller cells may occur as a result of increased release of these neurotransmitters from neurons, and decreased degradation. Quantification of the semisaturation value (half-maximal response) for glutamate and GABA Müller cell loading during postmortem ischemia indicated a shorter semisaturation value for GABA than glutamate. Such changes are consistent with a single aerobically dependent GABA-degradation pathway, and the existence of multiple glutamate-degradation pathways. Comparison with the in vitro ischemic model showed similar qualitative characteristics, but a markedly increased semisaturation time for glutamate and GABA Müller cell loading (a factor of 5-10) in the postmortem ischemia model. We interpret these differences to indicate that the in vitro condition provides a more immediate and/or severe ischemic insult. In the postmortem ischemia model, the delayed glial cell loading implies the availability of internal stores of both glucose and/or oxygen. Increased glial and neuronal immunoreactivity for the amino acids involved in transamination reactions, aspartate, alanine, leucine, and ornithine was observed, indicating a potential shift in the equilibrium of transamination reactions associated with glutamate production. These findings provide evidence that, in the rat retina, there are multiple pathways subserving glutamate production/degradation that include a multitude of transamination reactions. Further evidence is therefore provided to support a role for all four amino acids in glutamate metabolism within a variety of retinal neurons and glia.     

The scotopic threshold response of the cat ERG is suppressed selectively by GABA and glycine

Corneal electroretinograms (ERGs) were recorded from anesthetized cats under scotopic conditions. We examined whether the scotopic threshold response (STR) of the ERG could be functionally distinguished from scotopic PII and a-wave using intravitreal application of neuroactive agents. We found that neurotransmitters with active sites on third order neurons had several different effects. Results were: (1) glycine and gamma-amino butyric acid (GABA) selectivity suppressed the STR but had relatively small and/or opposite effects on PII; (2) serotonin, acetylcholine and dopamine were nonselective and suppressed both STR and PII; (3) strychnine blocked the suppression of the STR by glycine. GABA-a antagonists alone only partially blocked GABA effects on the STR, and GABA-b antagonists were ineffective; (4) strychnine enhanced the STR. Bicuculline also increased STR amplitudes, but only in the presence of haloperidol. Our results suggest that the retinal pathway that contributes to the rod-driven STR is strongly influenced by cells that release glycine or GABA in the dark. These cells are possibly third order neurons in the retina. Our results also suggest that picrotoxin and bicuculline can facilitate the release of dopamine in the cat retina. Furthermore, the data indicate a light evoked release of dopamine which was first noticeable at about two log units above ERG threshold.     

Retinal neurotransmission

The mammalian retina is classically divided into ten layers which contain the neuronal elements identified as photoreceptors, horizontal cells, bipolar cells, amacrine cells and ganglion cells. Using various neuroscientific techniques possible neurotransmitter substances have been assigned to each of these cell types. Thus the localization of transmitter synthesizing enzymes and storage vesicles, the demonstration of release of transmitter in response to specific stimuli, the observation of post-synaptic events mimicked or blocked by the iontophoretic application of exogenous transmitter/agonist or antagonist drug respectively, and the identification of efficient transmitter inactivation mechanisms synaptically add evidence for the association of certain proposed transmitter substances with specific neuronal elements. The evidence for the proposal that the excitatory amino acids glutamate and aspartate are transmitters of photoreceptors, that gamma-aminobutyric acid (GABA) is the inhibitory transmitter of horizontal and amacrine cells, that acetylcholine is associated with the functioning of bipolar cells, and that taurine, glycine and dopamine may all also play neurotransmitter or neuromodulatory roles at amacrine cell synapses is discussed.     

Immunohistochemical localization of amino acids in the diabetic retina of Goto-Kakizaki rats

The Goto-Kakizaki (GK) rat is a spontaneous model of non-insulin-dependent diabetes mellitus without obesity and diabetic retinopathy. We examined the retinal distribution of L-glutamate, gamma aminobutyric acid (GABA), glycine, and L-aspartate as neurotransmitters in the GK rat retina, using an immunohistochemical method with high-affinity antibodies. The retinal structures in the GK rats were the same as the controls. However, in the GK rats, immunoreactivity of L-glutamate and GABA was observed in the Müller and photoreceptor cells in addition to the immunoreactivity in normal rats. There was no change in glycine distribution between GK rats and controls. In the GK rats, L-aspartate accumulated in the inner segment of the photoreceptor cells in addition to the normal distribution. We consider that these immunoreactivity patterns in the GK rat retina might be induced by ischemia associated with diabetes mellitus.     

The action of inhibitory neurotransmitters, γ-aminobutyric acid and glycine may distinguish between the area centralis and the peripheral retina in cats

The effects of iontophoretically applied gamma-aminobutyric acid (GABA) and glycine, and of their antagonists, bicuculline and strychnine, were compared between ganglion cells from the central and peripheral retinae of optically intact eyes in barbiturate-anaesthetised cats. The visual response of on-cells was inhibited by GABA and enhanced by bicuculline. The visual response of off-cells was inhibited by glycine and enhanced by strychnine. The sensitivity of cells to the transmitters was lower in the peripheral retina than in the area centralis, whilst the sensitivity to the antagonists was similar in both regions of the retina. Cells from the area centralis were inhibited by either GABA or glycine, but never both. Cells from the periphery were less selective and were inhibited by both transmitters.     

Uptake of certain possible neurotransmitters into retinal neurons of some mammals

The uptake of glycine, GABA, and β-alanine was studied in rat, guinea-pig, cat, monkey and human retinas. The human retinas were studied in vitro only, the others both in vivo and in vitro.Either in vivo or in vitro [³H]glycine was aceumulated by certain cells which presumably are a type of amacrine cells. Glycine was not significantly metabolized under the conditions of the experiments. There was no significant variation between the species with this amino acid.[³H]GABA was preferentially accumulated by a type of amacrines in vivo. In rats and monkeys there was also a variable uptake into Müller cells already in vivo, and this uptake often disguised the neuronal uptake into amacrines. [³H]GABA in vitro gave predominantly glial uptake in all species except in cats and guinea-pigs where radioactive amacrines were still seen.[³H]β-alanine gave results similar to [³H]GABA.[³H]Glutamic acid produced predominantly glial uptake either in vivo or in vitro. Rods but not cones became somewhat radioactive in vitro.The results emphasize that particularly the GABA uptake varies significantly between different species.     

Factors affecting the spontaneous release of (3H)glycine from rabbit retina

The efflux of [3H]glycine was studied in superfused rabbit retina in the presence of various amino acids, ouabain, or high K+ or low Ca2+ concentrations in the superfusion medium. Unlabelled glycine evoked an accelerated efflux as did the structurally similar neutral alpha-amino acids. beta-alanine and GABA were ineffective. The results demonstrate a homoexchange of glycine, and a heteroexchange with the neutral alpha-amino acids. A low concentration of glutamic acid (10(-5) M) will release glycine from the retina. This is an ATPase dependent process which is partially blocked by a high Mg2+/Ca2+ ratio and which may be related to a retinal transmitter function of glutamic acid. A high concentration of K+ or the presence of ouabain in the superfusing medium greatly increases the rate at which glycine is lost from the retina.     

Glycine receptor subunit composition alters the action of GABA antagonists

GABA receptor antagonists produce an unexpectedly significant inhibition of native glycine receptors in retina and in alpha1 or alpha2 homomeric glycine receptors (GlyRs) expressed in HEK 293 cells. In this study we evaluate this phenomenon in heteromeric glycine receptors, formed by mixing alpha1, alpha2, and beta subunits. Picrotoxinin, picrotin, SR95531, and bicuculline are all more effective antagonists at GlyRs containing alpha2 subunits than alpha1 subunits. Inclusion of beta subunits reduces the inhibitory potency of picrotoxinin and picrotin but increases the potency of SR95531 and bicuculline. As a result of these two factors, bicuculline is particularly poor at discriminating GABA and glycine receptors. Picrotin, which has been reported to be inactive at GABA receptors, blocks glycine currents in retina and in HEK293 cells, suggesting its utility as a selective glycine antagonist. However, picrotin is a more potent inhibitor of GABA than glycine in retinal neurons. We also tested if GABA and glycine receptor subunits can combine to form functional receptors. If GABAAR gamma2S subunits are co-expressed with GlyR alpha subunits, the mixed receptor is glycine-sensitive and GABA-insensitive. But the mixed receptor exhibits a non-competitive picrotoxinin inhibition that is not observed in the homomeric GlyRs. This suggests that glycine and GABA subunits can co-assemble to form functional glycine receptors.     

Immunocytochemical localization of excitatory and inhibitory neurotransmitters in the zebrafish retina.

The patterns of glutamate, gamma-aminobutyric acid (GABA), and glycine distribution in the zebrafish retina were determined using immunocytochemical localization of antisera at the light-microscope level. The observed GABA immunoreactivity (GABA-IR) patterns were further characterized using antibodies to both isoforms of glutamic acid decarboxylase (GAD65 and GAD67), the synthetic enzyme for GABA. Glutamate-IR was observed in all retinal layers with photoreceptors, bipolar cells, and ganglion cells prominently labeled. Bipolar cells displayed the most intense glutamate-IR and bipolar cell axon terminals were clearly identified as puncta arranged in layers throughout the inner plexiform layer (IPL). These findings suggest the presence of multiple subtypes of presumed OFF- and ON-bipolar cells, including some ON-bipolar cells characterized by a single, large (9 microm X 6 microm) axon terminal. GABA-, GAD-, and glycine-IR were most intense in the inner retina. In general, the observed labeling patterns for GABA, GAD65, and GAD67 were similar. GABA- and GAD-IR were observed in a population of amacrine cells, a few cells in the ganglion cell layer, throughout the IPL, and in horizontal cells. In the IPL, both GABA- and GAD-IR structures were organized into two broad bands. Glycine-IR was observed in amacrine cells, interplexiform cells, and in both plexiform layers. Glycine-positive terminals were identified throughout the IPL, with a prominent band in sublamina 3 corresponding to an immunonegative region observed in sections stained for GAD and GABA. Our results show the distribution of neurons in the zebrafish retina that use glutamate, GABA, or glycine as their neurotransmitter. The observed distribution of neurotransmitters in the inner retina is consistent with previous studies of other vertebrates and suggests that the advantages of zebrafish for developmental studies may be exploited for retinal studies.     

Physiological and morphological properties of off- and on-center bipolar cells in the Xenopus retina: effects of glycine and GABA.

We studied the morphology and center-surround organization of Lucifer Yellow injected OFF- and ON-center bipolar cells in the light-adapted Xenopus retina and the effects of glycine and GABA on their cone-mediated light responses. In both classes of cell, prominent antagonistic surround responses up to 20 mV in amplitude could be evoked without first suppressing the center responses with steady illumination. An additional feature of the light-evoked bipolar cell response was a pronounced (up to -24 mV) delayed hyperpolarizing after potential (DHAP) which followed the depolarizing responses of both classes of bipolar cell. The morphological features of dye-injected bipolar cells conformed to the general idea of segregation of ON and OFF pathways in the inner and outer interplexiform layer, however, the morphology of axonal arborizations was different for both classes. OFF-center cells ramified symmetrically around the primary branchpoint, whereas ON-center cells had a strongly asymmetrical arrangement of their axonal tree. The center and surround responses were differentially sensitive to glycine and GABA. Glycine eliminated the antagonistic surround responses in both OFF and ON cells; the center responses were reduced to some extent but were not eliminated. In contrast, GABA affected the hyperpolarizing responses much more strongly than the depolarizing response components. That is, the amplitude of the center response in the OFF cell and the surround response in the ON cell was reduced 80-90% during exposure to GABA, whereas the surround and center depolarizations of OFF and ON cells, respectively, were reduced only 0-10%. Our findings implicate a role for GABAergic and glycinergic pathways in the center-surround organization of bipolar cells in Xenopus retina. In addition, the results suggest that the pathways mediating center-surround antagonism may be different in OFF-bipolar cells vs. ON-bipolar cells.     

Expression of GABA transporter subtypes (GAT1, GAT3) in the adult rabbit retina.

PURPOSE: GABA transporters (GATs) are of importance for GABA signal systems. They have previously not been examined in rabbit retina, nor has their correlation with neurotransmitter GABA and GABA receptors been examined in the retina of any species. METHODS: The distribution of GATs, GABA and GABA receptors was examined with immunohistochemical methods. RESULTS: Both GAT1 and GAT3 immunoreactivities were found in the inner plexiform layer and in amacrine cells. GAT3 was also present in Müller cells. GAT1 appeared in amacrine cells that also had a high GABA concentration, but not in cells with moderate to low GABA concentration. GAT1 was also present in amacrine cells that did not show GABA immunoreactivity, possibly indicating a postsynaptic GABA uptake system. CONCLUSION: GAT3 is probably involved in both neuronal and glial GABA uptake whereas GAT1 is involved in predominantly neuronal uptake, and possibly also into non-GABA-ergic amacrine cells. Further, there may be at least two populations of GABA containing neurons.     

Changes in localization of amino acids in the detached cat retina

PURPOSE: To investigate the distribution of amino acids (glutamate, aspartate, glutamine, GABA, glycine) in detached retinas with minimum postmortem artifact and to clarify the relation between amino acid distribution and histopathological change in the outer portion of detached retinas. METHODS: Unilateral retinal detachment was produced in cats by injecting 0.25% sodium hyaluronate into the subretinal space using a glass micropipet. The eyes were fixed by perfusion for 10 min, 1, 3, 6 and 24 h, 2, 3 and 7 days after detachment and then examined under conventional light- and electron-microscopic immunocytochemistry. RESULTS: For glutamate, aspartate and glutamine, the inner segments and perikarya of the photoreceptor cells, which were not immunopositive in the normal retinas, showed various degrees of immunoreactivity immediately after retinal detachment. Photoreceptor cells with the strong immunoreactivity developed necrosis. The staining pattern of GABA and glycine scarcely changed during the course of retinal detachment. CONCLUSIONS: Excess intracellular glutamate, aspartate and glutamine in photoreceptor cells may cause a part of neuronal death after retinal detachment.     

Glutamate and gamma-aminobutylic acid release into the vitreous during cataract surgery

OBJECTIVE: In cataract surgery, transient retinal ischemia and photostimulation-induced retinal damage occurs, even though only for a short duration. We suspected the possibility of excessive release of glutamate under these conditions, and therefore analyzed the amino acid contents in the vitreous body. MATERIALS AND METHODS: The vitreous bodies of 32 eyes with disease of the macular area necessitating retinovitreous surgery were used as samples for amino acid analysis. Ischemic diseases such as diabetic retinopathy, venous occlusion, and retinal detachment were excluded. The vitreous amino acid contents were compared between 18 eyes that underwent combined cataract and vitreous surgery and 14 eyes that had vitreous surgery alone. RESULTS: An approximately 3-fold increase of glutamate, 6-fold increase of gamma-aminobutylic acid (GABA), and 2-fold increase of alanine were observed in combined cataract and vitreous surgery compared to vitreous surgery alone. CONCLUSION: Even in human eyes, acute pressure overload or photostimulation for a short duration cause excessive release of glutamate. A simultaneous increase of GABA suggests the possibility that when glutamate is over-released, the retina physiologically reduced the concentration of glutamate via GABA, in an attempt to suppress the neurotoxin action.