Taurine, glutamate and GABA modulate the outgrowth from goldfish retinal explants and its concentrations are affected by the crush of the optic nerve

The amino acid taurine plays an important trophic role during development and regeneration of the central nervous system. Other amino acid systems, such as those for glutamate and gamma-aminobutyric acid (GABA), are modified during the same physiological and pathological processes. After crushing the optic nerve, goldfish retinal explants were plated in the absence and in the presence of different amino acids and amino acid receptor agonists. The length and the density of the neurites were measured at 5 days in culture. Taurine increased the length and the density of neurites. Glutamate and glycine increased them at low concentration, but were inhibitors at higher concentration. The combination of N-methyl-D-aspartate (NMDA) and glycine produced a greater inhibitory effect than NMDA alone. NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) added simultaneously with taurine impaired the stimulatory effect of the latter. GABA stimulated the emission of neurites in a concentration dependent manner. Hypotaurine also elevated the length of neurites, but cysteinsesulfinic acid did not produce a significant effect. The concentrations of taurine, glutamate and GABA were determined by HPLC with fluorescent detection in the retina of goldfish at various days post-crushing the optic nerve. The levels of taurine were significantly increased at 48h after the crush, and were elevated up to 20 days. Glutamate level decreased after the lesion of the optic nerve and was still low at 20 days. GABA concentration was not significantly different from the control. The interaction of these amino acids during the regenerative period, especially the balance between taurine and glutamate, may be a determinant in restoring vision after the crush.     

GABA and glutamate levels in the substantia nigra reticulata following repetitive cerebral ischemia in gerbils

Repetitive cerebral ischemia produces more severe damage than a similar single duration insult. We have previously shown that, in gerbils, damage in the substantia nigra reticulata (SNr) is seen with repetitive insults rather than a single insult. We have also shown that there is a progressive decrease in the extracellular GABA in the striatum in the days preceding such damage, speculating that a loss of GABA may be in part responsible for this damage. This study evaluates the GABA levels in the SNr in animals exposed to repetitive ischemic insults. Each animal received a total of three ischemic insults of 3-min duration at hourly intervals. In vivo microdialysis was carried out to analyze the GABA and glutamate dialysate levels on Days 1, 3, 5, 7, and 14 following the ischemic insult. In the control and treated (ischemic) animals, there was a significant increase in the GABA levels with the introduction of nipecotic acid on Days 1, 3, 5, and 14. However, on Day 7 there was a significant attenuation in the GABA response to nipecotic acid in the treated animals in comparison to the controls. The glutamate levels in the treated animals were similar to the control animals on Days 1, 3, 5, and 7. However, on Day 14 the glutamate levels were significantly lower than on previous days. Our experiments for the first time measure extracellular glutamate and GABA responses in the SNr in animals exposed to repetitive ischemic insults. Our experiments show that there is a significant decrease in the GABA concentrations at a time when ischemic damage is developing in this region. This confirms our hypothesis that a decrease in GABA may be one factor contributing to neuronal damage during the period following repetitive ischemic insults. Further, the rebound increase in GABA levels on Day 14 with a concomitant fall in glutamate levels would indicate that reparative processes are still active in the 2 weeks following the insult.     

The callosal connections of the hierarchically organized somatosensory areas of primates

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.     

In vivo injection of [1-13C]glucose and [1,2-13C]acetate combined with ex vivo 13C nuclear magnetic resonance spectroscopy: a novel approach to the study of middle cerebral artery occlusion in the rat

Astrocytes play a pivotal role in cerebral glutamate homeostasis. After 90 minutes of middle cerebral artery occlusion in the rat, the changes induced in neuronal and astrocytic metabolism and in the neuronal-astrocytic interactions were studied by combining in vivo injection of [1-13C]glucose and [1,2-13C]acetate with ex vivo 13C nuclear magnetic resonance spectroscopy and HPLC analysis of amino acids of the lateral caudoputamen and lower parietal cortex, representing the putative ischemic core, and the upper frontoparietal cortex, corresponding to the putative penumbra. In the putative ischemic core, evidence of compromised de novo glutamate synthesis located specifically in the glutamatergic neurons was detected, and a larger proportion of glutamate was derived from astrocytic glutamine. In the same region, pyruvate carboxylase activity, representing the anaplerotic pathway in the brain and exclusively located in astrocytes, was abolished. However, astrocytic glutamate uptake and conversion to glutamine took place, and cycling of intermediates in the astrocytic tricarboxylic acid cycle was elevated. In the putative penumbra, glutamate synthesis was improved compared with the ischemic core, the difference appeared to be brought on by better neuronal de novo glutamate synthesis, combined with normal levels of glutamate formed from astrocytic glutamine. In both ischemic regions, gamma-aminobutyric acid synthesis directly from glucose was reduced to about half, indicating impaired pyruvate dehydrogenase activity; still, gamma-aminobutyric acid reuptake and cycling was increased. The results obtained in the current study demonstrate that by combining in vivo injection of [1-13C]glucose and [1,2-13C]acetate with ex vivo 13C nuclear magnetic resonance spectroscopy, specific metabolic alterations in small regions within the rat brain suffering a focal ischemic lesion can be studied.     

Protein kinase inhibitors attenuate cardiac swelling-induced amino acid release in the rat

Rat Langendorff heart preparations have been used to study the efflux of cardiac amino acids into coronary artery perfusates during brief (5-min) periods of exposure to hyposmotic stress (70 mM NaCl). Coronary flow rates, heart rates and intra-aortic pressures were recorded. Amino acid levels were measured by high-performance liquid chromatography. Hyposmotic stress caused marked percentage increases in taurine, glutamate and aspartate levels in the coronary perfusate, with smaller increases in phosphoethanolamine, glycine and alanine and non-significant increases in serine and glutamine. Amino acid levels declined during reperfusion with isosmotic Krebs-Henseleit bicarbonate buffer. Inhibition of protein kinase C with chelerythrine chloride (5 microM) depressed the osmotically-induced release of aspartate, glutamate, taurine and glycine. The protein tyrosine kinase inhibitor, genistein, reduced the anisosmotic efflux of aspartate, glutamate, taurine and phosphoethanolamine. Lavendustin A, another inhibitor of tyrosine kinase, depressed the osmotically evoked release of aspartate, glutamate and taurine. These studies demonstrate the involvement of protein kinase C and tyrosine kinases in the efflux of amino acids from the osmotically challenged rat heart and imply that these enzymes are involved in the mechanisms responsible for volume regulation by cardiac cells.     

Maternal dietary protein deficiency decreases amino acid concentrations in fetal plasma and allantoic fluid of pigs

This study was conducted to test the hypothesis that maternal dietary protein deficiency decreases amino acid availability to the fetus, thereby contributing to retarded fetal growth. Primiparous gilts selected genetically for low or high plasma total cholesterol concentrations (low line and high line, respectively) were mated, and then fed 1.8 kg/d of isocaloric diets containing 13% or 0.5% crude protein. At d 40 or 60 of gestation, they were hysterectomized, and maternal and fetal blood samples as well as amniotic and allantoic fluids were obtained for analyses of amino acids, ammonia and urea. Dietary protein restriction decreased (P < 0.05) the following: 1) maternal plasma concentrations of urea at d 40 and 60 of gestation; 2) fetal plasma concentrations of alanine, arginine, branched-chain amino acids (BCAA), glutamine, glycine, lysine, ornithine, proline, taurine, threonine and urea at d 60 of gestation; 3) amniotic and allantoic fluid concentrations of urea at d 40 and 60 of gestation; and 4) allantoic fluid concentrations of alanine, arginine, BCAA, citrulline, cystine, glycine, histidine, methionine, proline, serine, taurine, threonine and tyrosine at d 40 of gestation, in gilts of both genetic lines. At d 60 of gestation, protein deficiency decreased (P < 0.05) allantoic fluid concentrations of arginine, cystine, glycine, taurine and tyrosine in low line gilts and of cystine, glutamine, ornithine, serine, taurine and tyrosine in high line gilts. Low line and high line gilts also differed remarkably in allantoic fluid concentrations of arginine, glutamine, ornithine and ammonia at d 40 and 60 of gestation. Our results suggest the following: 1) protein-deficient gilts maintain maternal plasma concentrations of amino acids by mobilizing maternal protein stores and decreasing oxidation of amino acids during the first half of gestation; 2) protein deficiency may impair placental transport of amino acids from the maternal to the fetal blood; and 3) low line and high line gilts differ in fetal amino acid metabolism. Decreases in concentrations of the essential and nonessential amino acids in the fetus may be a mechanism whereby maternal dietary protein restriction results in fetal growth retardation.     

Compartmentation of [14C]glutamate and [14C]glutamine oxidative metabolism in the rat hippocampus as determined by microdialysis

Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect 14CO2 generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced 14C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [14C]glutamate and [14C]glutamine were 59 +/- 18 and 2.1 +/- 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-14C]glutamate and [U-14C]glutamine were 408 +/- 8 and 387 +/- 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [14C]glutamine in the interstitial space when [14C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.     

Differential effects of unique profile antipsychotic drugs on extracellular amino acids in the ventral pallidum and globus pallidus of rats

The effects of antipsychotic drugs (APDs) on brain dopamine receptors in the striatum are ultimately expressed through efferent projections which primarily use amino acid transmitters, including gamma-aminobutyric acid and glutamate. The present study examined the effects of APDs on extracellular amino acid levels in the rat ventral pallidum (VP) and globus pallidus (GP), areas that receive projections from distinct striatal subregions. Clozapine, an APD with low motor side effect liability, and metoclopramide, a low-potency APD with high motor side effect liability, were compared with haloperidol, a widely used APD with high motor side effect liability. Drugs were administered subcutaneously and amino acid levels were monitored concurrently in the VP and GP by intracranial microdialysis. High doses of haloperidol and metoclopramide increased and clozapine decreased extracellular gamma-aminobutyric acid levels in the GP but not the VP. Low, but not high, doses of the three drugs tended to increase extracellular glutamate levels in both pallidal regions. Clozapine, but not the other two drugs, decreased extracellular threonine in the GP and glycine and threonine in the VP. Results indicate a correlation between increased gamma-aminobutyric acid levels in the GP and the propensity of the APDs tested to induce motor side effects. The novel effects of clozapine on extracellular glycine and threonine further distinguish this drug as a unique antipsychotic compound.     

Ontogeny of neurotransmitter amino acids in human fetal brains

A wide spectrum of developmental profiles is presented for a few important neurotransmitter amino acids, namely gamma-aminobutyric acid, glycine, glutamic acid, aspartic acid and taurine as well as the key enzymes involved in their metabolism in human fetal brains of different gestational ages. Besides taurine almost all these amino acids and their key metabolic enzymes were found to be progressively enhanced upto the mid period of third trimester of pregnancy indicating a rapid growth of nerve processes, myelination and maturation of fetal brains particularly during this period. Interestingly taurine showed an inverse relationship of ontogenic pattern with respect to its biosynthesizing enzyme in fetal brains.     

Extracellular amino acid levels in the interpositus nucleus during classical eyeblink conditioning in alert cats.

The extracellular levels of selected amino acids in the cerebellar posterior interpositus nucleus (PIN) during classical eyeblink conditioning was analyzed in alert cats using a delay paradigm. Animals were prepared for the chronic recording of eyelid movements (with the magnetic search-coil technique) and the electromyographic activity of the orbicularis oculi muscle. With the help of a guide and push-pull cannulae, selected PIN sites were perfused daily during classical eyeblink conditioning. The perfusate was sampled at intervals of 5 min and analyzed with a high-pressure liquid chromatography- electrochemical detection (HPLC-EC) method. The analysis of push-pull perfusate revealed a significant increase in the release of glycine, taurine, and glutamate across the successive conditioning sessions, in parallel with the acquisition of eyelid conditioned responses (CRs). Both CRs and extracellular levels of these three amino acids returned to control values during extinction. Other amino acids (alanine, GABA, glutamine, serine, and threonine) did not undergo modifications in their extracellular concentrations across the training. Results are discussed with regard to the role of PIN in this type of associative learning. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Potassium-evoked efflux of transmitter amino acids and purines from rat cerebral cortex

Repeated applications of elevated K+ (50 or 75 mM) in cerebral cortical cup superfusates was used to evoke an efflux of gamma-aminobutyric acid (GABA), glutamate, aspartate, glycine, adenosine, and inosine from the in vivo rat cerebral cortex. K+ (50 mM) significantly elevated GABA levels in cup superfusates but had little effect on the efflux of glutamate, aspartate, glycine, adenosine, or inosine. K+ (75 mM) significantly enhanced the efflux of GABA, aspartate, adenosine, and inosine and caused nonsignificant increases in glutamate and glycine efflux. The adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA), applied in cup superfusates at a concentration of 10(-10) M had no effect on either basal or K(+)-evoked release of any of the amino acids or purines measured. At 10(-6) M CPA significantly enhanced aspartate release, and depressed GABA efflux. The selective A2 adenosine receptor agonist 2-p(2-carboxyethyl) phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS 21680) (10(-8) M) was without effect on either basal, or K(+)-evoked, efflux of amino acids or purines. The enhancement of aspartate (an excitotoxic amino acid) efflux by higher concentrations of CPA is likely due to activation of adenosine A2b receptors. This observation may be of relevance when selecting adenosinergic agents to treat ischemic or traumatic brain injuries. Overall, the results suggest that effects of adenosine receptor agonists on K(+)-evoked efflux of transmitter amino acids from the in vivo rat cerebral cortex may not be comparable to those observed with in vitro preparations.     

Selective alterations of extracellular brain amino acids in relation to function in experimental portal-systemic encephalopathy: results of an in vivo microdialysis study

Portal-systemic encephalopathy (PSE) is characterized by neuropsychiatric symptoms progressing through stupor and coma. Previous studies in human autopsy tissue and in experimental animal models of PSE suggest that alterations in levels of brain amino acids may play a role in the pathogenesis of PSE. To assess this possibility, levels of amino acids were measured using in vivo cerebral microdialysis in frontal cortex of portacaval-shunted rats administered ammonium acetate (3.85 mmol/kg, i.p.) to precipitate severe PSE. Sham-operated rats served as controls. Portacaval shunting resulted in significant increases of levels of extracellular glutamine (threefold, p < 0.001), alanine (38%, p < 0.01), aspartate (44%, p < 0.05), phenylalanine (170%, p < 0.001), tyrosine (140%, p < 0.001), tryptophan (63%, p < 0.001), leucine (75%, p < 0.001), and serine (60%, p < 0.001). Administration of ammonium acetate to sham-operated animals led to a significant increase in extracellular glutamine and taurine content, but this response was absent in shunted rats. The lack of taurine release into extracellular fluid following ammonium acetate administration in portacaval-shunted rats could relate to the phenomenon of brain edema in these animals. Ammonium acetate administration resulted in significant increases in the extracellular concentrations of phenylalanine and tyrosine in both sham-operated and portacaval-shunted rats. Severe PSE was not accompanied by significant increases in extracellular fluid concentrations of glutamate, aspartate, GABA, tryptophan, leucine, or serine, suggesting that increased spontaneous release of these amino acids in cerebral cortex is not implicated in the pathogenesis of hepatic coma.     

Glutamate, glutamine, and GABA as substrates for the neuronal and glial compartments after focal cerebral ischemia in rats

BACKGROUND AND PURPOSE: Even though the utilization of substrates alternative to glucose may play an important role in the survival of brain cells under ischemic conditions, evidence on changes in substrate selection by the adult brain in vivo during ischemic episodes remains very limited. This study investigates the utilization of glutamate, glutamine, and GABA as fuel by the neuronal and glial tricarboxylic acid cycles of both cerebral hemispheres after partially reversible focal cerebral ischemia (FCI). METHODS: Right hemisphere infarct was induced in adult Long-Evans rats by permanent occlusion of the right middle cerebral artery and transitory occlusion of both common carotid arteries. (1,2-13C2) acetate was infused for 60 minutes in the right carotid artery immediately after carotid recirculation had been re-established (1-hour group) or 23 hours later (24-hour group). Extracts from both cerebral hemispheres were prepared and analyzed separately by 13C nuclear magnetic resonance and computer-assisted metabolic modeling. RESULTS: FCI decreased the oxidative metabolism of glucose in the brain in a time-dependent manner. Reduced glucose oxidation was compensated for by increased oxidations of (13C) glutamate and (13C) GABA in the astrocytes of the ipsilateral hemispheres of both groups. Increased oxidative metabolism of (13C) glutamine in the neurons was favored by increased activity of the neuronal pyruvate recycling system in the 24-hour group. CONCLUSIONS: Data were obtained consistent with time-dependent changes in the utilization of glutamate and GABA or glutamine as metabolic substrates for the glial or neuronal compartments of rat brain after FCI.     

Longitudinal changes of brain amino acid content occurring before, during and after epileptic activity

In cats affected with cortical epileptogenic foci induced by penicillin application to and cobalt implantation into the pericruciate area, the brain amino acids contents were determined in the focus as well as in extrafocal areas. In different groups of animals, brain removal for biochemical determinations was performed at different times before, during and after epilepsy and the values compared to controls. The only amino acid to show a significant change before appearance of spikes in both types of epilepsy was taurine, which decreased. Cobalt epilepsy was accompanied by changes in a larger number of amino acids than penicillin epilepsy: in the former the brain content of taurine, GABA, aspartate, glutamate, serine, threonine, glycine and alanine was altered. The changes were proportional to the severity of epilepsy and more prominent in the focus area. After disappearance of spikes the levels of most amino acids returned to normal except for some amino acids, previously unaffected by penicillin epilepsy, which were decreased. It is proposed that the decrease in brain taurine, occurring before the appearance of penicillin and cobalt epilepsy, could increase the excitability of a certain neuronal population and thus, by potentiating the effects on neurons of penicillin and cobalt, contribute to the initiation of epilepsy.     

Organic osmolytes in the kidney of domesticated red deer, Cervus elaphus

The cortex, inner and outer medulla, and papilla of kidneys of domestic red deer were analysed. In hydrated animals the urine concentration was found to be 672 +/- 45 mOsm.l-1. The medullary and papillary regions of the kidney were rich in the osmolytes betaine, glycerophosphorylcholine (GPC), inositol and sorbitol, all of which showed a steep rise in concentration from cortex to papilla. The kidney was rich in free amino acids, in particular taurine, glutamate (+glutamine), glycine and alanine, which were present at concentrations sufficient to suggest a possible role as osmolytes.     

Age-specific distribution of plasma amino acid concentrations in a healthy pediatric population

Reference values were determined for 23 plasma free amino acids from measurements done in 148 healthy children ranging from 0 to 18 years of age. Amino acid analysis was performed by ion-exchange chromatography. We propose a graphic form of presenting the age-specific distribution of plasma amino acid concentrations where the 10th, 50th, and 90th quantiles are illustrated. Although each amino acid possesses its own pattern of distribution, we can identify five different profiles. Nine amino acids (alanine, arginine, asparagine, methionine, ornithine, phenylalanine, proline, threonine, and tyrosine) demonstrate a decrease in their concentrations during the first year of life; their concentrations then tend to increase throughout childhood and adolescence. Nine others (cystine, glutamine, glycine, histidine, isoleucine, leucine, lysine, tryptophan, and valine) show a steady increase throughout infancy, childhood, and adolescence. Five amino acids (aspartic acid, citrulline, glutamic acid, serine, and taurine) do not follow these two common profiles. For the first time, quantile curves are produced to illustrate the age-dependent variation of amino acid concentrations from infancy to adulthood. This alternative way of presenting amino acid concentrations may facilitate the follow-up of patients with inborn errors of amino acid metabolism.     

Neuroprotective effects of lamotrigine in global ischemia in gerbils. A histological, in vivo microdialysis and behavioral study

A sudden surge in the release of glutamate is currently believed to be an important initiating step in neuronal damage due to an ischemic insult. In this experiment, we tested the efficacy of neuroprotection with lamotrigine, a novel antiepileptic drug that blocks voltage gated sodium channels and inhibits the ischemia-induced release of glutamate in the gerbil forebrain model of cerebral ischemia. The medication was administered 30 min before and 30 min after the insult in two groups of animals. Histological assessment of neuronal damage was evaluated at 7 and 28 days after the ischemic insult. Animals evaluated at 28 days also underwent behavioral testing. Microdialysis was used in the same model to study the response of ischemia-induced glutamate in saline treated controls versus animals treated with lamotrigine 20 min before the insult. There was highly significant neuronal protection in animals who were treated with lamotrigine either before or after the insult. Protection was seen both at 7 and 28 days after the insult. Behavioral testing also showed significantly better recovery in both sets of animals in comparison to the saline-treated group. Microdialysis confirmed a significant attenuation of the ischemia-induced glutamate surge when compared to the saline-treated animals. Our morphological, behavioral and microdialysis experiments show that lamotrigine offers significant neuroprotection from the effects of transient forebrain ischemia in gerbils. Neuroprotection with post-ischemic therapy probably depends on preserving the capacity of the sodium/calcium exchanger to reduce intracellular calcium concentrations or persistent 'toxicity' of glutamate in the reperfusion period on the already 'primed' injured neurons. These concepts need further study.     

Comparison of the actions of glycine and related amino acids on isolated third order neurons from the tiger salamander retina

Whole cell voltage and current clamp recordings were obtained from third order neurons isolated from the salamander retina. Using cross desensitization, the structure-function relationship of short chain amino acids on the glycine receptor were examined. L-Serine, L-alanine, beta-alanine and taurine all cross desensitized with glycine, but did not show significant cross desensitization with GABA. This indicates that these amino acids act at the glycine receptor. The order of potency was glycine > beta-alanine > taurine > L-alanine > L-serine. TAG, a reputed selective taurine antagonist, was equally effective in blocking taurine and glycine currents. There is no evidence for distinct receptors for taurine. Amino acids with larger moieties at the alpha carbon, such as threonine and valine, produced inactive ligands. Placing a methyl group on the amine of glycine or esterification of the carboxyl group also greatly reduced activity. Based on these modifications of the glycine molecule, it appears that selectivity at the glycine receptor results in part from steric restrictions at all three sites in the glycine chain. The steric interference is most critical at the carboxyl and amino ends, and less limiting at the alpha carbon. Doses of L-serine that had only slight effects in voltage clamp experiments, nevertheless produced large effects in current clamp experiments. This indicates that several endogenous amino acids can have significant effects on membrane voltage, even when their shunting activity may be small. High concentrations of agonists produced desensitization in the voltage clamp records, but there was little evidence of desensitization in the current clamp experiments. These results indicate that several endogenous amino acids can activate the glycine receptor, but there is no evidence for a discrete receptor for taurine, beta-alanine, L-alanine or L-serine. Since all these endogenous amino acids have similar amino and acid terminals, reduction in potency results from steric interference around the alpha carbon. This graded potency may have functional significance in mediating inhibition.     

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.     

Uric acid protects neurons against excitotoxic and metabolic insults in cell culture, and against focal ischemic brain injury in vivo

Uric acid is a well-known natural antioxidant present in fluids and tissues throughout the body. Oxyradical production and cellular calcium overload are believed to contribute to the damage and death of neurons that occurs following cerebral ischemia in victims of stroke. We now report that uric acid protects cultured rat hippocampal neurons against cell death induced by insults relevant to the pathogenesis of cerebral ischemia, including exposure to the excitatory amino acid glutamate and the metabolic poison cyanide. Confocal laser scanning microscope analyses showed that uric acid suppresses the accumulation of reactive oxygen species (hydrogen peroxide and peroxynitrite), and lipid peroxidation, associated with each insult. Mitochondrial function was compromised by the excitotoxic and metabolic insults, and was preserved in neurons treated with uric acid. Delayed elevations of intracellular free calcium levels induced by glutamate and cyanide were significantly attenuated in neurons treated with uric acid. These data demonstrate a neuroprotective action of uric acid that involves suppression of oxyradical accumulation, stabilization of calcium homeostasis, and preservation of mitochondrial function. Administration of uric acid to adult rats either 24 hr prior to middle cerebral artery occlusion (62.5 mg uric acid/kg, intraperitoneally) or 1 hr following reperfusion (16 mg uric acid/kg, intravenously) resulted in a highly significant reduction in ischemic damage to cerebral cortex and striatum, and improved behavioral outcome. These findings support a central role for oxyradicals in excitotoxic and ischemic neuronal injury, and suggest a potential therapeutic use for uric acid in ischemic stroke and related neurodegenerative conditions.