Using the rd1 mouse to understand functional and anatomical retinal remodelling and treatment implications in retinitis pigmentosa: A review.

Retinitis Pigmentosa (RP) reflects a range of inherited retinal disorders which involve photoreceptor degeneration and retinal pigmented epithelium dysfunction. Despite the multitude of genetic mutations being associated with the RP phenotype, the clinical and functional manifestations of the disease remain the same: nyctalopia, visual field constriction (tunnel vision), photopsias and pigment proliferation. In this review, we describe the typical clinical phenotype of human RP and review the anatomical and functional remodelling which occurs in RP determined from studies in the rd/rd (rd1) mouse. We also review studies that report a slowing down or show an acceleration of retinal degeneration and finally we provide insights on the impact retinal remodelling may have in vision restoration strategies.

Glutamate uptake in retinal glial cells during diabetes.

AIMS: Glutamate recycling is a major function of retinal Muller 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 Muller 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 Muller cells of diabetic rats compared to controls (p<0.001). The EC(50) was found to increase by 0.304 log units in diabetic Muller 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 Muller 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.

Localization of amino acid neurotransmitters following in vitro ischemia and anoxia in the rat retina.

Glutamate and gamma-aminobutyric acid (GABA) are two of the dominant neurotransmitters in the retina and brain. The production/degradation of glutamate and GABA involves an intricate interrelationship between neurons and glia, as well as aerobic and anaerobic metabolic pathways. The aim of this work was to develop an in vitro model of retinal ischemia/anoxia and determine the changes in cellular localization of glutamate and GABA and the time course for such changes. After anoxic/ischemic insult, glutamate and GABA rapidly accumulate within glia with GABA showing a quicker time course and larger magnitude change. The accumulation time constant for both glutamate and GABA under anoxic conditions was dependent upon glucose concentration: high glucose levels resulted in delayed glial amino acid loading. The differences in time constants between GABA and glutamate glial loading most likely reflect the multitude of glutamate degradation pathways compared to the single aerobically dependent GABA pathway. Oxygen availability and reduced glucose (hypoglycemia) lead to an almost immediate increase (within 1 min) of glutamate and GABA labelling within glia. In addition, altered labelling patterns were found under anoxic/ischemic conditions for amino acids involved in glutamate transamination reactions: aspartate, leucine, alanine. and ornithine. These changes are consistent with alterations of equilibria of enzymatic reactions involved in glutamate metabolism, and thus support a role for all four amino acids in glutamate metabolism within a variety of retinal neurons.

Cellular diversity in mouse neocortex revealed by multispectral analysis of amino acid immunoreactivity.

Cortical cells were classified using an unsupervised cluster analysis based upon their quantitative and combinatorial immunoreactivity for glutamate, gamma-aminobutyric acid (GABA), aspartate, glutamine and taurine. Overall, cell class-specific amino acid signatures were found for 12 cellular types; seven GABA-immunoreactive (GABA-IR) populations (GABA1--7), three classes containing high glutamate levels (GLUT1--3) and two putative glial (GLIA1, 2) cell types. From their large somata, associated vertical processes and high glutamate content, the GLUT classes most probably correspond to pyramidal neurons. Two of the GLUT classes demonstrated complementary distributions in different cortical layers, suggesting spatial separation of cells differing in amino acid immunoreactivity. Of the seven GABA classes, two comprised cells with large somata and displayed medium to low glutamate levels. On the basis of size, these two populations may correspond to large basket cell interneurons. Glial populations could be divided into two classes: GLIA1 cells were more frequently associated with blood vessels and GLIA2 cells were more commonly seen in the lower cortical layers. This work demonstrates that signature recognition based upon amino acid content can be used to separate cortical cells into different categories and reveal further subclasses within these categories. This approach is complementary to other methods using physiological and molecular tools and ultimately will enhance our understanding of neuronal heterogeneity.

Word acuity threshold as a function of contrast and retinal eccentricity.

BACKGROUND: We determined word acuity thresholds as a function of contrast and retinal eccentricity to determine the rate of threshold alteration in the normal retinal periphery. METHODS: Subjects identified words presented foveally (0 degrees eccentricity) or above the point of fixation at retinal eccentricities of 0.5, 1, 2, 3, 6, and 8 degrees for three contrast levels of 10, 45, and 85%. A descending method of limits was used to determine thresholds for random four-letter words flashed for 90 ms at the different retinal eccentricities. RESULTS: For high-contrast letters, word acuity displayed threshold elevation in the periphery similar to previous reports and similar threshold elevation to those reported for vernier acuity. Lower contrast levels displayed different threshold change as a function of eccentricity, approaching levels reported for grating acuity. When comparing the relative elevation of word acuity thresholds for the different contrast levels (85 vs. 45% and 85 vs. 10%), both comparisons showed that the most rapid decline in word acuity threshold occurs within 2 degrees of the fovea. CONCLUSIONS: The peripheral retina displays a reduction in word acuity threshold that is dependent on letter contrast and shows a change similar to those reported for higher cortical functions such as vernier thresholds. The greatest word threshold elevation occurs within the central 2 degrees of the fovea.

Characterisation of dark adaptation in human cone pathways: an application of the equivalent background hypothesis.

It is well accepted that in rod photoreceptors the photoproducts generated by a bleach cause desensitisation during dark adaptation. We examine whether this notion holds for cones. A model of cone dark adaptation is developed based on the equivalent background concept. The underlying theory of the model relies on a series of assumptions that link psychophysically determined detection thresholds to cone phototransduction. Correction of thresholds for the reduced quantum-catching ability of the cones (due to the depletion of photopigment caused by a bleaching light) is an important aspect of the model. Foveal detection thresholds were measured for a small test flash presented on a large steady background field or presented alone after adapting to the background field. Test and background fields were monochromatic, with wavelengths closely matched to promote detection by the luminance mechanism. The model provided a good description of the data collected under these conditions. Parameters of the model were similar for all wavelengths and each observer, as were the derived equivalent background relationships. Analysis of previously published data for Stiles' pi5 mechanism gave analogous results. The model is made up of two components. The early (fast) component is likely to be due to the direct action of the cone equivalent of inactivated Rh* on the G-protein cascade and/or the reverse reaction of the cone equivalent of inactivated Rh* to Rh*. The later (slow) component may be due to the direct action of cone opsin on the G-protein cascade.

Glutamate, GABA and precursor amino acids in adult mouse neocortex: cellular diversity revealed by quantitative immunocytochemistry.

Glutamate is an important amino acid in the neocortex for metabolic and neurotransmitter functions. The objective of this study was to detect variations in cellular glutamate content using quantitative immunocytochemistry. We show that glutamate is present in almost all cortical cells and coexists with other amino acids such as aspartate, glutamine or gamma-aminobutyric acid (GABA). The patterns of aspartate and glutamine content suggests that there are no purely aspartatergic or glutaminergic neurons. GABAergic neurons showed variable levels of the precursors such as glutamate, glutamine and aspartate. Comparison of immunoreactive patterns between two cortical areas did not detect any statistically significant differences. The mean cellular intensity for GABA and glutamate was constant across different layers. Surprisingly, we found that GABAergic neurons could coexist with either low or high levels of glutamate, suggesting that metabolic levels of glutamate in these neurons could be variable. Alternatively, some GABA neurons may utilize both GABA and glutamate for neurotransmission. We show that when variations in amino acid content are separately mapped onto individual cells, co-registration is a useful technique for reporting heterogeneity among cortical cells.

Endogenous IGF-1 regulates the neuronal differentiation of adult stem cells.

Stem cells from the adult forebrain of mice were stimulated to form clones in vitro using fibroblast growth factor-2 (FGF-2). At concentrations above 10 ng/ml of FGF-2, very few clones gave rise to neurons; however, if FGF-2 was removed after 5 days, 20-30% of clones subsequently gave rise to neurons. The number of neuron-containing clones and the number of neurons per clone was significantly enhanced, if insulin-like growth factor (IGF)-1 or heparin were added subsequent to FGF-2 removal. The spontaneous production of neurons after FGF-2 removal was shown to be due to endogenous IGF-1, since antibodies to IGF-1 and an IGF-1 binding protein totally inhibited neuronal production. Similarly, these reagents also abrogated the neuron-promoting effects of heparin. Thus, it appears that endogenous IGF-1 may be a major regulator of stem cell differentiation into neurons. Furthermore, it was found that high levels of IGF-1 or insulin promoted the maturation and affected the neurotransmitter phenotype of the neurons generated.

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.

Amino acid neurochemistry of the vertebrate retina.

The dominant neurochemicals involved in encoding sensory information are the amino acid neurotransmitters, glutamate, gamma-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.

Reduced glutamate uptake by retinal glial cells under ischemic/hypoxic conditions.

The high-affinity uptake of glutamate by glial cells and neurons of the central nervous system, including the retina, serves to inactivate synaptically released glutamate and maintains glutamate at low concentrations in the extracellular space. This uptake prevents accumulation of glutamate extracellularly and thus minimizes the possibility of glutamate neurotoxicity secondary to ischemic insult. One mechanism whereby glutamate neurotoxicity may occur in ischemic/hypoxic insult is through increased extracellular K+ reversing the electrogenic glutamate uptake into retinal glial (Müller) cells. We investigated glial uptake of the amino acids glutamate, GABA, and D-aspartate in the intact isolated rat retina under high extracellular K+ conditions and under conditions simulating ischemia. Immunocytochemical findings showed that uptake of glutamate and GABA by MIller cells in the intact isolated rat retina continues under conditions simulating ischemia and high extracellular K+ conditions, and uptake of D-aspartate also continues under high K+ conditions. However, under high K+ conditions, the glutamate uptake system saturates at a lower concentration of exogenous glutamate than in the normal K+ condition. These findings provide evidence that disruption of glutamate uptake by Müller cells is likely to be a significant contributing factor to excess glutamate accumulation in the extracellular space which can lead to neurotoxicity.

Separate progenitors for radial and tangential cell dispersion during development of the cerebral neocortex.

Cell lineage analyses suggest that cortical neuroblasts are capable of undertaking both radial and tangential modes of cell movement. However, it is unclear whether distinct progenitors are committed to generating neuroblasts that disperse exclusively in either radial or tangential directions. Using highly unbalanced mouse stem cell chimeras, we have identified certain progenitors that are committed to one mode of cell dispersion only. Radially dispersed neurons expressed glutamate, the neurochemical signature of excitatory pyramidal cells. In contrast, tangential progenitors gave rise to widely scattered neurons that are predominantly GABAergic. These results suggest lineage-based mechanisms for early specification of certain progenitors to distinct dispersion pathways and neuronal phenotypes.

Amino acid signatures in the normal cat retina.

PURPOSE: To establish a nomogram of amino acid signatures in normal neurons, glia, and retinal pigment epithelium (RPE) of the cat retina, guided by the premise that micromolecular signatures reflect cellular identity and metabolic integrity. The long-range objective was to provide techniques to detect subtle aberrations in cellular metabolism engendered by model interventions such as focal retinal detachment. METHODS: High-performance immunochemical mapping, image registration, and quantitative pattern recognition were combined to analyze the amino acid contents of virtually all cell types in serial 200-nm sections of normal cat retina. RESULTS: The cellular cohorts of the cat retina formed 14 separable biochemical theme classes. The photoreceptor --> bipolar cell --> ganglion cell pathway was composed of six classes, each possessing a characteristic glutamate signature. Amacrine cells could be grouped into two glycine- and three gamma-aminobutyric acid (GABA)-dominated populations. Horizontal cells possessed a distinctive GABA-rich signature completely separate from that of amacrine cells. A stable taurine-glutamine signature defined Müller cells, and a broad-spectrum aspartate-glutamate-taurine-glutamine signature was present in the normal RPE. CONCLUSIONS: In this study, basic micromolecular signatures were established for cat retina, and multiple metabolic subtypes were identified for each neurochemical class. It was shown that virtually all neuronal space can be accounted for by cells bearing characteristic glutamate, GABA, or glycine signatures. The resultant signature matrix constitutes a nomogram for assessing cellular responses to experimental challenges in disease models.

Characteristics of anisometropic suppression: simple reaction time measurements.

The characteristics of artificially induced anisometropic suppression were investigated in observers with normal and abnormal binocular vision (anisometropic amblyopia) by using a simple reaction time paradigm. Reaction time was measured as a function of stimulus intensity for various stimulus durations. For all conditions, the reaction time increased as stimulus intensity decreased toward threshold. We found that traditional techniques for modeling this trend were inadequate, so we developed a simple visuogram method for comparing these functions. Using this technique, reaction time versus intensity functions are shown to be shape-invariant for all conditions examined. This means that, although reaction times are longer during induced anisometropic suppression or in anisometropic amblyopia, they are the same if contrast is normalized to equate threshold. The shape-invariant nature of these functions is also consistent with the notion that a single mechanism mediates detection under these conditions. Temporal summation was investigated at both threshold (method of limits) and suprathreshold (criterion reaction time) levels. Again, because of shape invariance, the suprathreshold results mirror the threshold results. The critical duration (the duration at the intersection of the complete summation and zero summation regions) is not affected by any of the conditions. However, the critical intensity (the intensity for the zero summation region) is higher for the amblyopic eyes, as compared with the normal or nonamblyopic eyes. Induced anisometropic suppression always increases the critical intensity, with a smaller increase occurring for the amblyopic eyes. This suggests that amblyopic eyes do not have a need for strong suppression.

Neurochemical development of the degenerating rat retina.

The Royal College of Surgeons' (RCS) rat is an experimental model for a group of hereditary retinal diseases commonly called retinitis pigmentosa. We used postembedding immunocytochemistry to determine the localisation of glutamate, gamma-aminobutyric acid (GABA), glycine, aspartate, glutamine, taurine, and arginine in the RCS rat retina during postnatal development. In addition, we evaluated the uptake characteristics for the three dominant amino acid neurotransmitters, glutamate, GABA, and glycine. Whereas, cellular localisation of all amino acids was similar to control retinas, there were major changes in the level of immunoreactivity, even before eye opening, and well before the onset of visibly detectable photoreceptor degeneration. Two major patterns emerged. First, neurochemical changes evident before degeneration, involving the amino acids glutamate, GABA, aspartate, glutamine, and arginine. Second, neurochemical changes that become evident during photoreceptor degeneration involving the amino acids taurine and glycine. Anomalies in uptake characteristics also become evident during the degeneration phase and are likely to reflect changes in cellular function as a consequence of the degeneration process. Neurochemical changes evident before photoreceptor degeneration involve both glutamate and GABA manufacturing pathways. Müller's cells displayed elevated levels of glutamine and arginine from an early age, and the neuroblastic layer in the RCS retina showed high glutamate levels. Modified aspartate immunoreactivity began at postnatal day 11 and is consistent with altered metabolic activity. These results suggest that amino acid neurochemistry is different in the RCS rat retina from an early age, which may indicate an underlying metabolic defect affecting multiple cell classes.

Localisation of amino acid neurotransmitters during postnatal development of the rat retina.

We used postembedding immunocytochemistry to determine the localisation of the amino acid neurotransmitters glutamate, gamma-aminobutyrate (GABA), and glycine, potential neurotransmitter precursors (aspartate and glutamine), and taurine in the rat retina during postnatal development. All amino acids investigated were present at birth; however, only the inhibitory neurotransmitters GABA and glycine displayed neuronal localisation. GABA was localised in a sparse population of amacrine cells, and glycine immunoreactivity was found in cells within the ventricular zone that appeared to migrate through the neuroblastic layer. Glutamate labelling was diffuse across the retina until postnatal day (PND) 8. Localisation of glutamine was evident within Müller's cells by PND 6, in agreement with the known age of onset of glutamine synthetase activity. Based on the findings of uptake of radiolabelled glutamate and GABA by PND 8 and changes in immunoreactivity, we propose that Müller's cells evolve at PND 6-8 from their precursor cells, the radial glial cells. Evidence for differences in glutamate turnover in the infant retina was seen on examination of aspartate and glutamine immunoreactivity. Aspartate labelling was weak until PND 11, when ganglion cells and some amacrine cells were labelled. Unlike the mature retina, a large number of amacrine cells were glutamine immunoreactive in the PND 6 retina. One reason for the observed differences in precursor pooling may be a lack of neuronal neurotransmitter release and overall low metabolic activity. We also investigated the response of the developing retina to ischaemic insult to test the physiological hypoxia model of vascular development. Our findings are consistent with the hypothesis that the developing retina has increased tolerance to ischaemic insult. Our findings suggest that, although the retina is morphologically adult like by PND 8, there are differences in neurotransmitter turnover in the immature rat retina.

L and M cone input into spectral sensitivity functions: a reanalysis.

In order to better understand the nature of long-wavelength (L) and middle-wavelength (M) cone input into spectral sensitivity functions and determine the reliability with which it is possible to predict L:M cone inputs, we developed analytical methods to determine confidence intervals for L:M cone input for spectral sensitivity functions or data transformed to cone-contrast space. Spectral sensitivity functions measured by direct heterochromatic brightness matches are dominated by the L/M opponent channel over most of the spectral range. For detection of large/ long test stimuli, spectral sensitivity functions show a characteristic "notch" at the adapting wavelength, with the L/M opponent channel dominating most of the spectral range. Flicker increment threshold (FIT) spectral sensitivity functions display many of the characteristics of the luminance flicker mechanism described by Stromeyer et al. (1987). [Vision Research, 27, 1113-1137]. Previous modelling of FIT spectral sensitivity functions proposed a 2:1 L:M cone input for most of testing conditions. We show that FIT spectral sensitivity functions are dominated by L cones but show L cone suppression under bright red adapting fields. For the fitted spectral sensitivity functions or simulated data sets, we found small confidence intervals for L:M cone input into the L/M opponent channel and conclude that it is possible to reliably predict L:M cone input ratios. However, for similar data sets of additive spectral sensitivity functions, we found large confidence intervals for L:M cone input ratios and conclude that it is not possible reliably predict L:M cone input into the L/M non-opponent channel using available spectral sensitivity functions.

Neurochemical architecture of the normal and degenerating rat retina.

We used post-embedding immunocytochemistry to determine the cellular localization of glutamate, gamma-amino butyric acid (GABA), glycine, aspartate, glutamine, arginine, and taurine in the normal and degenerating rat retina. Müller's cell function was also evaluated by determining the uptake and degradation characteristics for glutamate. Immunocytochemical localization of amino acids in adult Royal College of Surgeons (RCS) and control rat retinas were similar with respect to cell classes. Differences in the intensity of labelling for glutamate, aspartate, glutamine, and glycine were observed in several classes of neurons, but the most prominent differences were shown by bipolar cells of the adult RCS rat retina. In addition, glutamine labelling within Müller's cells was higher in the RCS rat than the control. These changes may have occurred because of alterations in the glutamate production or degradation pathways. We tested this hypothesis by determining Müller's cells glutamate uptake and degradation characteristics in adult and postnatal day 16 RCS retinas. High affinity uptake of 3[H]-glutamate revealed an accumulation of grains over Müller's cell bodies in the adult RCS retina implying glutamate degradation anomalies. We confirmed anomalies in glutamate metabolism in RCS Müller's cells by showing that exogenously applied glutamate was degraded over a longer time course in postnatal day 16 RCS retinas, compared to control retinas. Differences in arginine immunoreactivity in adult and immature RCS retinas conform to the presumed dysfunction of Müller's cells in these degenerating retinas. The anomalies of amino acid localization, uptake and degradation lead us to conclude that Müller's cells in the RCS retina show abnormal function by postnatal day 16; an earlier time to previously reported anatomical and functional changes in this animal model of retinal degeneration.

Amino acid signatures in the primate retina.

Pattern recognition of amino acid signals partitions virtually all of the macaque retina into 16 separable biochemical theme classes, some further divisible by additional criteria. The photoreceptor-->bipolar cell-->ganglion cell pathway is composed of six separable theme classes, each possessing a characteristic glutamate signature. Neuronal aspartate and glutamine levels are always positively correlated with glutamate signals, implying that they largely represent glutamate precursor pools. Amacrine cells may be parsed into four glycine-dominated (including one glycine/GABA immunoreactive population) and four GABA-dominated populations. Horizontal cells in central retina possess a distinctive GABA signature, although their GABA content is constitutively lower than that of amacrine cells and shows both regional and sample variability. Finally, a taurine-glutamine signature defines Müller's cells. We thus have established the fundamental biochemical signatures of the primate retina along with multiple metabolic subtypes for each neurochemical class and demonstrated that virtually all neuronal space can be accounted for by cells bearing characteristic glutamate, GABA, or glycine signatures.

Glutamate metabolic pathways in displaced ganglion cells of the chicken retina.

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.