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.     

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.     

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.     

Amino acid immunoreactivity in normal human retina and after brachytherapy

We localised amino acids in the mid‐peripheral aged human retina and a retina that had undergone radiation treatment 10 years earlier. The distribution pattern of glutamate, γ‐amino butyric acid (GABA), glycine, glutamine and taurine, reflected patterns established in the primate retina. The retina that had undergone radiation exposure displayed both anatomical and neurochemical remodelling. The proximal retina comprised around 40 to 45 per cent of the total retina and neuronal kinesis and aberrant neuronal projections were also present. Amino acid neurochemistry was strikingly different with Müller cells displaying GABA loading, glycinergic neurons displaced and displaying a very high level of glycine labelling. We conclude that radiation exposure triggered these changes in the human retina and likely reflects general remodelling of structure and function following ischaemic damage to endothelial cells.     

Localization of NMDA receptor subunits and mapping NMDA drive within the mammalian retina

Glutamate is a major neurotransmitter in the retina and other parts of the central nervous system, exerting its influence through ionotropic and metabotropic receptors. One ionotropic receptor, the N-methyl-D-aspartate(NMDA) receptor, is central to neural shaping, but also plays a major role during neuronal development and in disease processes. We studied the distribution pattern of different subunits of the NMDA receptor within the rat retina including quantifying the pattern of labelling for all the NRI splice variants, the NR2A and NR2B subunits.The labelling pattern for the subunits was confined predominantly in the outer two-thirds of the inner plexiform layer. We also wanted to probe NMDA receptor function using an organic cation, agmatine (AGB); a marker for cation channel activity. Although there was an NMDA concentration-dependent increase in AGB labelling of amacrine cells and ganglion cells, we found no evidence of functional NMDA receptors on horizontal cells in the peripheral rabbit retina, nor in the visual streak where the type A horizontal cell was identified by GABA labelling. Basal AGB labelling within depolarizing bipolar cells was also noted. This basal bipolar cell AGB labelling was not modulated by NMDA and was completely abolished by the use of L-2-amino-4-phosphono-butyric acid,which is known to hyperpolarize retinal depolarizing bipolar cells. AGB is therefore not only useful as a probe of ligand-gated drive, but can also identify neurons that have constitutively open cationic channels. In combination,the NMDA receptor subunit distribution pattern and the AGB gating experiments strongly suggests that this ionotropic glutamate receptor is functional in the cone-driven pathway of the inner retina.     

兔眼视网膜下注射N-甲基右旋天冬氨酸诱导视网膜变性的组织病理学研究

目的研究视网膜下注射兴奋性氨基酸N-甲基右旋天冬氨酸（NMDA）对神经细胞变性的作用。方法取12只1个月龄有色家兔,视网膜下注射10μl（30mmol／L）NMDA（溶剂为DMEM-F12）,形成视网膜隆起,在注射12、24、48h及1周后分别处死家兔,取其视网膜组织进行免疫组织化学检测和电镜观察。应用抗Calretinin、Calbindin、PKCα抗体,分别标记视网膜无长突细胞、水平细胞及视杆双极细胞;采用原位缺口末端标记技术（TUNEL）技术标记凋亡细胞。结果损伤早期（12～24h）,实验组视网膜可见散在细胞核固缩浓染的光感受器细胞,并有无长突细胞和神经节细胞的早期严重变性;中期（48h）视网膜各层神经元均出现病理性改变;损伤晚期（1周）视网膜各层细胞数目明显减少。损伤早期,TUNEL技术标记的阳性细胞位于视网膜各层。免疫组织化学和形态学计量资料显示视网膜下注射NMDA后,水平细胞、无长突细胞及神经节细胞数目明显减少,视杆双极细胞数目基本无变化。超微结构观察显示有凋亡、坏死、水肿变性及混合型细胞死亡等多种变性形式。结论视网膜下聚集NMDA时,光感受器细胞、水平细胞、视杆双极细胞、无长突细胞及神经节细胞均表现为视网膜兴奋性毒性反应,与以往体内及体外研究结果显示的仅有内核层神经元死亡情况不同。     

Inner retinal mechanisms engaged by retinal electrical stimulation

PURPOSE: Retinal prosthetic devices are being developed to bypass degenerated retinal photoreceptors by directly activating retinal neurons with electrical stimulation. However, little is known about retinal activity during such stimulation. METHODS: Whole cell patch-clamp recordings were obtained from ganglion and bipolar cells in the salamander retinal slice preparation. A stimulating electrode was positioned at the vitreal surface of the slice. RESULTS: Brief pulses of cathodic current evoked transient inward currents in ganglion cells arising from action potentials. Longer pulses (>5 milliseconds) also evoked sustained inward currents in ganglion cells that appeared synaptic in origin because, unlike transient currents, sustained currents were blocked by inhibiting synaptic transmission with Cd2+. These synaptic currents reversed around ECl and were blocked by picrotoxin, strychnine, or both, suggesting they were mediated by GABAa/c and glycine receptors. Synaptic currents were also blocked by the NMDA antagonist MK801 and the KA/AMPA antagonist NBQX, suggesting that epiretinal stimulation evoked glutamate release from bipolar cells, which in turn stimulated the release of GABA and glycine from amacrine cells. Sustained currents were also evoked by epiretinal stimulation in bipolar cells. These currents reversed near ECl and were blocked by picrotoxin, suggesting they arose from GABAa/c receptors. CONCLUSIONS: Pulse duration is an important parameter for effective activation of the inner retina by epiretinal stimulation. Brief pulses evoke action potentials only in ganglion cells. However, longer pulses also evoke sustained synaptic currents by stimulating glutamate release from bipolar cell terminals, which, in turn, evokes the release of GABA and glycine from amacrine cells.     

Anatomical and electrophysiological evidence for GABAergic bipolar cells in tiger salamander retina

Our previous work showed that about 12% of bipolar cells in salamander retina synthesize and take up gamma-aminobutyric acid (GABA), are GABA transporter (GAT)-immunoreactive, and respond with a GAT current to extracellularly applied GABA, suggesting that these bipolar cells use GABA, in addition to glutamate, as a neurotransmitter. Further support for this idea was obtained in this study by use of immunogold electron microscopy and whole-cell patch clamp electrophysiology. Ultrastructural analysis showed that amacrine cell and ganglion cell processes were postsynaptic to GABA-immunoreactive synapses made by bipolar cell axon terminals. Whole-cell recordings were obtained from amacrine and ganglion cells in response to activation of bipolar cells by puffing KCl at their dendrites in the outer plexiform layer. Inhibitory postsynaptic currents were observed in several third order neurons, even after blocking the excitatory postsynaptic responses, generated in the inner plexiform layer, with a combined application of NMDA and non-NMDA receptor antagonists, AP-5 and CNQX. These ultrastructural and electrophysiological data support our previous neurochemical results, and suggest that the retinal through-information pathway in salamander includes both inhibitory GABAergic as well as excitatory glutamatergic synaptic mechanisms.     

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.     

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.     

Inner and outer retinal mechanisms engaged by epiretinal stimulation in normal and rd mice

Retinal prosthetic devices are being developed to bypass degenerated retinal photoreceptors by directly activating retinal neurons with electrical stimulation. However, the retinal circuitry that is activated by epiretinal stimulation is not well characterized. Whole-cell patch clamp recordings were obtained from ganglion cells in normal and rd mice using flat-mount and retinal slice preparations. A stimulating electrode was positioned along the ganglion cell side of the preparation at different distances from the stimulated tissue. Pulses of cathodic current evoked action potentials in ganglion cells and less frequently evoked sustained inward currents that appeared synaptic in origin. Sustained currents reversed around E(Cl) and were inhibited by blockade of α-amino-3-hydroxyl-5-methyl-4-isoxazole-proprionate (AMPA)-type glutamate receptors with 2,3-dihydroxy-6-nitro-sulfamoyl-benzo(f)-quinoxaline-2,3-dione (NBQX), γ aminobutyric acid a/c (GABA(a/c)) receptors with picrotoxinin, or glycine receptors with strychnine. This suggests that epiretinal stimulation activates glutamate release from bipolar cell terminals, which in turn evokes release of GABA and glycine from amacrine cells. Synaptic current thresholds were lower in ON ganglion cells than OFF cells, but the modest difference did not attain statistical significance. Synaptic currents were rarely observed in rd mice lacking photoreceptors compared to normal retina. In addition, confocal calcium imaging experiments in normal mice retina slices revealed that epiretinal stimulation evoked calcium increases in the outer plexiform layer. These results imply a contribution from photoreceptor inputs to the synaptic currents observed in ganglion cells. The paucity of synaptic responses in rd mice retina slices suggests that it is better to target retinal ganglion cells directly rather than to attempt to engage the inner retinal circuitry.     

The beta-adrenoceptor antagonists metipranolol and timolol are retinal neuroprotectants: comparison with betaxolol

beta-adrenoceptor antagonists are used clinically to reduce elevated intraocular pressure in glaucoma which is characterised by a loss of retinal ganglion cells. Previous studies have shown that the beta(1)-selective adrenoceptor antagonist, betaxolol, is additionally able to protect retinal neurones in vitro and ganglion cells in vivo from the detrimental effects of either ischemia-reperfusion or from excitotoxicity, after topical application. The neuroprotective effect of betaxolol is thought not to be elicited through an interaction with beta-adrenoceptors, but by its ability to reduce influx of sodium and calcium through voltage-sensitive calcium and sodium channels.In the present study it is shown that the non-selective beta-adrenoceptor antagonists, metipranolol and timolol behave like betaxolol. When topically applied they all attenuate the detrimental effect of ischemia-reperfusion. Protection of the retina was determined by evaluating changes in the electroretinogram and by assessing the loss of mRNA for Thy-1, which is expressed in retinal ganglion cells. In addition, studies conducted on neurones in mixed retinal cultures demonstrated that metipranolol, betaxolol and timolol were all able to partially counteract anoxia-induced cell loss and viability reduction. The influence of timolol was, however, not significant. Within the confines of these investigations, an order of neuroprotective efficacy was delineated for the three beta-adrenoceptor antagonists: betaxolol>metipranolol>timolol. The ability of the beta-adrenoceptor antagonists to attenuate ligand-induced stimulation of calcium and sodium entry into neuronal preparations showed a similar order of effectiveness.In conclusion, the ability to confer neuroprotection to retinal neurones is a common feature of three ophthalmic beta-adrenoceptor antagonists (betaxolol, metipranolol and timolol). A comparison of the effectiveness of the individual compounds in protecting retinal cells in vivo was not possible in these studies. However, in vitro studies show that the capacity of the individual beta-adrenoceptor antagonists to act as neuroprotectants appears to relate to their capacity to attenuate neuronal calcium and sodium influx.     

Comparison of the neuroprotective effects of adrenoceptor drugs in retinal cell culture and intact retina

PURPOSE: The efficacy of beta1-adrenoceptor (AR)-selective (betaxolol and metoprolol) and nonselective (timolol) antagonists and the alpha2-AR agonist UK14,304 as retinal neuroprotectants was compared and contrasted in an in vitro glutamate excitotoxicity model. The ability of UK14,304, brimonidine, and betaxolol to alter glutamate-receptor-induced changes in intracellular calcium ([Ca2+]i) was also determined in isolated retinal neurons and retinal ganglion cells (RGCs) in an intact retina preparation. METHODS: Neuronal survival was measured in mixed retinal cell cultures treated for 24 hours with media containing 100 microM glutamate, with or without the addition of each of the drugs (1-1000 microM). Effects of glutamate on glia were also investigated in a C6 glioma cell line. Glutamate-induced changes in [Ca2+]i with and without UK14,304, and its analogue brimonidine were assessed by calcium-imaging techniques in retinal neurons in culture. The effect of betaxolol on [Ca2+]i was investigated in RGCs in intact rabbit retina. RESULTS: In cell cultures, 10-1000 microM glutamate resulted in a dose-dependent loss of neurons, but not of glia. The absence of glutamate toxicity in glia was confirmed in C6 glioma cells. Betaxolol, but not timolol or metoprolol, significantly increased survival (from 52% of control in glutamate-only to 78% with 10 microM betaxolol) after excitotoxic insult. UK14,304 also increased survival (from 62% of control in glutamate only to 109% and 101% of control with 10 and 100 microM UK14,304, respectively). This effect was blocked by the specific alpha2-antagonist, yohimbine. Both UK14,304 and brimonidine (10-100 microM) reduced glutamate-induced [Ca2+]i increases in retinal neurons in culture. The actions of the alpha2-agonists in reducing glutamate-induced [Ca2+]i increases were reduced by yohimbine (1 microM). Betaxolol (100 microM) reduced N-methyl-D-aspartate (NMDA)-induced increases of [Ca2+]i in RGCs in intact retina. CONCLUSIONS: Betaxolol reduced glutamate excitotoxicity in retinal neurons in vitro through a mechanism independent of beta-AR interactions. UK14,304, acting through alpha2-ARs, was also neuroprotective in vitro. The neuroprotective actions of betaxolol and the alpha2-agonists on retinal neurons may be due, at least in part, to a direct reduction of glutamate receptor-mediated increases of [Ca2+]i.     

Expression and distribution of ionotropic glutamate receptor subunits on parasol ganglion cells in the primate retina

The response properties of postreceptoral sensory neurones are determined by the properties of their input neurones, by intrinsic membrane properties, and by the properties of neurotransmitter receptors on the soma and dendritic tree. We previously showed that inhibitory neurotransmitter (GABA(A) and glycine) receptors on a well-characterised sensory neurone, the parasol ganglion cell in the primate retina, are segregated towards the distal part of the dendritic tree. Here we studied the distribution of excitatory ionotropic glutamate receptor subunits on the dendrites of parasol cells in the retina of a New World monkey, the marmoset, Callithrix jacchus. Individual ganglion cells were intracellularly injected in an in vitro retinal wholemount preparation. Ionotropic glutamate receptor subunits. including AMPA (GluR1-4), kainate (GluR6/7), NMDA (NR1C2') subunits, and the orphan receptors delta1 and delta2 were visualized with immunocytochemical methods. Immunoreactive puncta that colocalized with the dendrites of ganglion cells were analyzed using standard and/or confocal light microscopy. Colocalized puncta were present on parasol dendrites for all subunits studied, but their density was much lower (approximately 1/5) than previously reported for inhibitory (GABA and glycine) receptors. Segregation of the glutamate receptor clusters (GluR1, GluR6/7 subunits) to the peripheral dendrites was less marked than that shown for GABA and glycine receptor clusters. No sign of segregation of colocalized puncta to the peripheral part of the dendritic field was seen with antibodies to the GluR2, GluR2/3, GluR4, delta1/2, or NR1C2' subunits. The results suggest that although there is diverse expression of glutamate receptor subtypes, the glutamatergic synapses form only a small proportion of the total synaptic input to primate ganglion cells. They further suggest that the processes which control distribution of excitatory and inhibitory synapses on the dendritic field of ganglion cells are, at least to some extent, independent.     

Most calretinin-containing amacrine cells in the rabbit retina co-localize glycine

Calretinin-containing retinal amacrine cells are heterogeneous with regard to their neurochemical properties. In the rabbit retina, about 90% of them contain glycine, as evidenced in the present study by double-label immunocytochemistry. In a previous report, we showed that a small population of amacrine cells contains both gamma-aminobutyric acid and calretinin. In this study, we further identified this cell population by means of known secondary markers. However, none of the markers we tested (choline acetyltransferase, serotonin accumulation, NADPH-diaphorase, vasoactive intestinal polypeptide) co-localized with calretinin. A small population (1%) of the cells in the ganglion cell layer contains both calretinin and glycine. Since calretinin-positive cells in the ganglion cell layer have been identified as ganglion cells based on soma size and presence of calretinin-positive axons in the optic nerve fiber layer, this population may represent a class of ganglion cell which contains glycine. Our results, together with those of other studies, suggest that calretinin is not a general marker of any of the well-known amacrine cell types in the mammalian retina. Rather, calretinin, just as other calcium-binding proteins, is distributed in a species-specific manner. At the same time it appears that, as shown for horizontal cells, one or more of the major buffer-type calcium-binding proteins of the EF-hand family is present in most of the retinal amacrine cells.     

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.     

Quantification and characterization of GABA‐ergic amacrine cells in the retina of GAD67‐GFP knock‐in mice

Purpose: Although the presence of γ‐aminobutyrate acid (GABA) in amacrine cells and its co‐localization with other neuronal substances is well known, there exists only little information about their quantitative distribution in the mouse eye. The aim of the present study was to characterize GABA‐ergic amacrine cells in the retina of the recently introduced glutamate decarboxylase 67‐green fluorescent protein (GAD67‐GFP) knock‐in mouse. Methods: Whole mounts of the retina were prepared and the GFP‐positive neurons quantified. Immunofluorescence staining was performed with antibodies against GABA, calbindin (CB), calretinin (CR), parvalbumin (PV), choline acetyl transferase (ChAT), tyrosine hydroxylase (TH), vesicular glutamate transporter (VGluT) 1, VGluT2 and VGluT3. Results: Displaced GABA‐ergic amacrine cells in the ganglion cell layer (GCL) showed a density of 1006 ± 170 cells/mm². In the inner nuclear layer (INL), the density of amacrine cells was 8821 ± 448 cells/mm² in the central region and 6825 ± 408 cells/mm² in the peripheral region. GFP‐positive amacrine cells co‐localized with GABA (99%), CR (INL 18%, GCL 71.3%), CB (INL 6.3%), bNOS (INL 1%, GCL 4%), and ChAT (INL 17%, GCL 92.6%). No co‐localization was seen with antibodies against PV, TH, and VGluT 1‐3. Conclusions: This study presents the first quantitative data concerning the co‐localization of GABA‐ergic neurons in the mouse retina with various neuronal markers.     

Interrelationship between retinal ischaemic damage and turnover and metabolism of putative amino acid neurotransmitters,glutamate and GABA

Conditions causing a reduction of oxygen availability (anoxia), such as stroke or diabetes, result in drastic changes in ion movements, levels of neurotransmitters and metabolites and subsequent neural death. Currently, there is no clinically available treatment for anoxia induced neural cell death resulting in drastic and permanent central nervous system dysfunction. However, there have been some exciting developments in experimentally induced anoxic conditions where several classes of drugs appear to significantly reduce neural cell death. This report aims to provide the foundations for understanding both the basic mechanisms involved in retinal ischaemic damage and experimental treatments used to prevent such damage. We discuss the normal release, actions and uptake of the fast retinal neurotransmitters, glutamate and GABA, in the vertebrate retina. Immunocytochemistry is used to demonstrate that both glutamate and GABA are found in the macaque retina. Following this is a discussion on how ischaemia may enhance neurotransmitter release or disrupt its uptake, thus causing an increase in extracellular concentration of these neurotransmitters and subsequent neuronal damage. The mechanisms involved in glutamate neurotoxicity are reviewed, because excess glutamate is the likely cause of retinal ischaemic damage. Finally, the mechanisms behind four possible modes of treatment of neurotransmitter toxicity and their advantages and disadvantages are discussed. Hopefully, further research in this area will lead to the development of a rational therapy for retinal, as well as cerebral ischaemia.Abbreviations -KG -ketoglutarate - AAT aspartate amino transferase - AC amacrine cell - ACL amacrine cell layer - BC bipolar cell - CNS central nervous system - EAA excitatory amino acids - G'ase glutaminase - GABA -amino butyric acid - GABA-T GABA transaminase - GAD glumatic acid decarboxylase - GC ganglion cell - GCL ganglion cell layer - GDH glutamate dehydrogenase - gj gap junction - GS glutamine synthetase - HC horizontal cell - ILM inner limiting membrane - INL inner nuclear layer - IPC inter-plexiform cell - IPL inner plexiform layer - IS inner segment of photoreceptor - NFL nerve fibre layer - NMDA N-methyl-D-aspartate - OLM outer limiting membrane - ONL outer nuclear layer - OPL outer plexiform layer - OS outer segment of photoreceptor - Ox acetateoxaloacetate - RL receptor layer - SSAD succinate semi-aldehyde decarboxylase - Succinate SA succinate semi aldehyde - TCA cycle tricarboxylic acid cycle     

Susceptibilities to and mechanisms of excitotoxic cell death of adult mouse inner retinal neurons in dissociated culture

PURPOSE: To explore the susceptibilities of adult retinal neurons in dissociated culture to treatments with excitotoxic agonists and the mechanisms of the resultant retinal cell death. METHODS: C57B6 mice were used. Retinas were removed, dissociated, plated on a polylysine/laminin substrate, and maintained in vitro for 5 to 7 days. Excitotoxic agonists (glutamate, N-methyl-D-aspartate [NMDA], or kainic acid [KA]) were added for 30 minutes or 24 hours, sometimes in the presence of modified extracellular ion concentrations or potential blocking agents. The next day, cells were fixed and immunocytochemically stained to identify ganglion and amacrine cells. Surviving cells were counted. RESULTS: Ganglion cells from adult mouse retinas were much less susceptible to excitotoxic death than those prepared from neonatal retinas. Adult amacrine cells were killed by KA, NMDA, or glutamate. Experiments with selective blockers demonstrated that KA killed through AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, whereas NMDA and glutamate exerted toxicity through a combination of AMPA and NMDA receptors. The KA-induced death of amacrine cells was not mediated by chloride ions. Removal of extracellular sodium, however, completely prevented the amacrine cell death, and removal of extracellular calcium prevented approximately 70% of the death. The path of calcium entry was investigated. Experiments with selective blockers indicated that the lethal calcium entry was via reverse operation of a sodium-calcium exchanger. CONCLUSIONS: There is a profound developmental regulation in the sensitivity of retina ganglion cells to excitotoxic insults. Excessive intracellular sodium and calcium are the proximal causes of amacrine cell death. The pathologic calcium entry is dependent on the sodium overload, which then drives a sodium-calcium exchanger to take up calcium.