Regional alterations in brain amino acids during the estrous cycle of the rat

Concentrations of 11 amino acids, including the neurotransmitters GABA, glutamate, aspartate, glycine and taurine, were determined in 12 brain regions of female rats during different stages of the estrous cycle. In addition, amino acids and sex hormone levels were determined in plasma. All sample collections were done in the forenoon between 9 and 11 a.m. Most regional amino acid levels measured did not change signficantly during estrous cycle, but significant alterations were found for GABA and glutamate in hypothalamus. Both amino acids were slightly decreased in hypothalamus during proestrus, which might reflect an alteration of GABA turnover in response to the high estrogen levels during this stage. A decreased glutamate level during proestrus was also found in thalamus, while both glutamate and GABA did not vary throughout estrous cycle in any of the other examined regions, including substantia nigra, amygdala, striatum, cortex and hippocampus. When diestrus was subdivided according to progesterone levels, high levels of this hormone seemed to be associated with effects on metabolism of certain amino acids, including glycine in substantia nigra, alanine in thalamus and threonine in pons/medulla. However, the few changes in regional amino acid concentrations found during the estrous cycle were so small that the functional significance of these changes cannot be ascertained without further determination of the cellular or subcellular compartments of brain tissue involved.     

Amino Acids in the Substantia Nigra of Rats with Striatal Lesions Produced by Kainic Acid

In an attempt to estimate the pool size of glutamate and other amino acids in γ-aminobutyric acid (GABA)-containing neurons, we determined the content of 12 amino acids in the bilateral substantia nigra of rats, in which unilateral striatal lesions had been made with kainic acid two weeks earlier. The assay of the amino acids (including glutamate, aspartate, glutamine, asparagine, glycine, and GABA) and ethanolamine was based on HPLC and fluorimetric detection after precolumn derivatization with o-phthaldialdehyde. The levels of all measured amino acids (except those of tyrosine, threonine, and ethanolamine) were decreased in the affected striatum, but only the levels of aspartate, taurine, and GABA were lowered in the ipsilateral substantia nigra. These results indicate that the pool size of the various amino acids in the striatonigral GABAergic pathway is small compared to their nigral content, and that in addition to GABA a significant fraction of aspartate and taurine may be confined to nerve terminals in the substantia nigra.     

The use of microdialysis for studying the regional effects of pharmacological manipulation on extracellular levels of amino acids--some methodological aspects.

The purpose of this study was to examine and validate the use of microdialysis for sampling and pharmacologically manipulating extracellular amino acids in the brain. Repeated use of microdialysis probes in acute intracerebral experiments did not significantly alter the relative recovery in vitro for the amino acids quantitated (GABA, aspartate, glutamate, glycine, taurine, and alanine). Regional differences in basal levels of some of the amino acids were detected in dialysates collected from the dorsomedial hypothalamus, striatum, and frontal cortex. The percent in vitro recoveries for the amino acids from the probes used in the three regions were not significantly different suggesting that the regional differences in basal levels of amino acids were functionally derived and not a consequence of variations in probe recovery. Perfusion with nipecotic acid, an inhibitor of GABA uptake, resulted in selective elevations in extracellular GABA in the three regions studied. Conversely, perfusion with high-potassium, a depolarizing agent, resulted in significant elevations in not only extracellular GABA but also aspartate, glutamate, and taurine. Thus, microdialysis is a method which can be employed to assess and to pharmacologically manipulate extracellular amino acids in the rat brain.     

Changes in free amino acids in the brain during embryonic development in layer and broiler chickens

Developmental changes in the levels of the excitatory amino acids l-glutamate (Glu) and l-Aspartate (Asp) and inhibitory amino acids glycine (Gly) and γ-amino butyric acid (GABA), as well as taurine and its related amino acids l-methionine (Met), l-cysteine (Cys) and l-serine (Ser) in the brain and pectoralis muscle at various embryonic stages and hatch in broiler and layer type chickens were determined. Brain concentrations of Asp, GABA and taurine were higher than those in the muscle, but the difference in the two types was small. The concentrations of the precursors of taurine including Met, Cys and Ser were lower than that of taurine. In conclusion, the synthesis of some amino acids and their metabolites such as Asp, GABA and taurine in the chick embryo is very high in order to support brain development.     

The free amino acids in peripheral nerves and in isolated inhibitory and excitatory nerve fibres of Cancer magister

Abstract— Free amino acids in whole nerve and in excitatory and inhibitory fibres isolated from the walking legs of the crab, Cancer mgister, have been determined, using a densitometric method which permits quantitative estimates of 1 nmol of a ninhydrin-positive substance on paper chromatograms. In confirmation of previous reports, whole nerve contained 5 amino acids at levels greater than 10 mmol/kg wet wt., including two anions (glutamate and aspartate) of major importance. The concentrations of 12 other amino acids were also estimated. The free amino acid fraction contributed about 40% (415 mosmol/kg of cell water) to the osmotic concentration of the tissue. Isolated inhibitory fibres were distinguished by a 15-fold higher level of GABA than that found in excitatory fibres (46 vs 3·1 mmol/l. of axoplasm). The level of proline also differed in the two types of fibres, but in contrast to GABA it was less concentrated in inhibitory fibres. As a consequence of these and of other smaller differences, the total concentration of free amino acids was slightly less in inhibitory fibres (365 vs 405 mmol/l. of axoplasm in excitatory fibres). The demonstration of the presence of GABA in crab inhibitory axons supports earlier suggestions that it may be the inhibitory transmitter at crab neuromuscular junctions.     

Amino Acid Content of Nerve Endings (Synaptosomes) in Different Regions of Brain: Effects of Gabaculine and Isonicotinic Acid Hydrazide

Abstract: The amino acid content of synaptosomes was determined in six regions of rat brain, and in all regions the five predominant amino acids were glutamate, glutamine, aspartate, taurine, and GABA (γ-aminobutyrate). However, the proportions of the individual amino acids varied considerably from one region to another, the GABA content being particularly high and the taurine content low in synaptosomes from the diencephalon and mesencephalon. Administration of isonicotinic acid hydrazide to rats lowered the synaptosomal GABA level by similar amounts in all brain regions, but the administration of gabaculine resulted in a particularly long-acting elevation in GABA levels in the nerve endings of the diencephalon and mesencephalon. The possibility is raised that the high GABA levels in the nerve terminals of the diencephalon may be involved in the gabaculine-induced lowering of the body temperature of the rats. A constancy in the amount of the synaptosomal pool of "aspartate + glutamate + glutamine + GABA" was observed despite large changes in the relative amounts of the four amino acids brought about by gabaculine.     

Evidence using in vivo microdialysis that aminotransferase activities are important in the regulation of the pools of transmitter amino acids

The effect of aminooxyacetic acid (AOAA), an inhibitor of pyridoxal phosphate-dependent enzymes (including the aminotransferases), on the K⁺-evoked release of amino acids was studied during microdialysis of neostriatum in anesthetized rats. K⁺-evoked (100 mM) release of asparatate, glutamate, and GABA was inhibited by 74%, 70%, and 63%, respectively, by 20 mM Mg²⁺ and are therefore reflecting release from the transmitter pools of these amino acids. Treatment with AOAA decreased the K⁺-evoked release of aspartate, glutamate, and GABA instantly, with a delayed decrease in the efflux of glutamine and alanine, arguing that the synthesis of transmitter amino acids in particular is sensitive to the activity of pyridoxal phosphate-dependent enzymes. Interestingly, GABA release increased severalfold following the initial decrease, probably reflecting inhibition by AOAA on GABA aminotransferase, the enzyme most sensitive to inhibition by AOAA, and responsible for enzymatic inactivation of transmitter GABA.Special issue dedicated to Dr. Claude Baxter.     

In Vivo Studies on the Extracellular, and Veratrine-Releasable, Pools of Endogenous Amino Acids in the Rat Striatum: Effects of Corticostriatal Deafferentation and Kainic Acid Lesion

The effects of corticostriatal deafferentation (decortication) and destruction of intrinsic neurons (intrastriatal kainate injection) on the extracellular concentration, and veratrine-releasable pools, of endogenous amino acids in the rat striatum were examined using the in vivo brain dialysis technique. Intracellular amino acid content was also determined. Decortication reduced selectively intra- and extracellular levels of glutamate (Glu) and aspartate (Asp). Extracellular changes were more pronounced than those in tissue content. gamma-Aminobutyric acid (GABA), taurine (Tau), and phosphoethanolamine (PEA) levels were not affected, whereas nonneuroactive amino acids were increased at 1 week but not at 1 month post-lesion. The intracellular pool of Glu and Asp was also reduced in kainate-lesioned striata. However, extracellular levels of these compounds were not affected significantly by this treatment. The tissue content of all other amino acids was decreased, the most prominent change being in the concentration of GABA. Extracellular GABA concentration was also reduced dramatically, whereas the concentrations of noneuroactive amino acids were increased to varying degrees. These data suggest that transmitter pools of neuroactive amino acids are an important supply for their extracellular pools. Lesion-induced alterations in nonneuroactive amino acids are discussed with regard to the loss of metabolic pools, glial reactivity, and changes in blood-brain barrier transport. Veratrine induced a massive release of neuroactive amino acids such as Glu, Asp, GABA, and Tau into the extracellular fluid, and a delayed increase in PEA. Extracellular levels of neuroactive amino acids were raised slightly. Decortication reduced, selectively, the amounts of Glu and Asp released by veratrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-dependence of the evoked release from guinea pig cerebral cortex slices of endogenous 14C-labelled amino acids, labelled via D-[U-14C]glucose.

Spontaneous and electrically evoked release of exogenous labelled amino acids and endogenous amino acids labelled from D-[U-14C]glucose were compared in control and Ca2+-free medium using guinea pig cerebral cortex slices. Spontaneous release of all labelled amino acids, except that of endogenous 14C-labelled threonine-serine-glutamine (unseparated) and exogenous [14C]aspartate, was doubled in Ca2+-free medium. The major portion of the electrically evoked release of endogenous [14C]glutamate, [14C]aspartate, gamma-amino[14C]butyrate (14C-labelled GABA) and exogenous 3H-labelled GABA was Ca2+-inpendent. More than half of the evoked release of the other labelled amino acids was Ca2+-independent. As the pattern of Ca2+-dependence of the evoked release concurred with the selectivity of the evoked release for endogenous [14C]-glutamate, [14C]aspartate, and 14C-labelled GABA, it was concluded that these labelled amino acids were probably released from the amino acid 'transmitter pool'.     

Taurine and GABA release from mouse cerebral cortex slices: Effects of structural analogues and drugs

The effects of structural analogues, excitatory amino acids and certain drugs on spontaneous and potassium-stimulated exogenous taurine and GABA release were investigated in mouse cerebral cortex slices using a superfusion system. Spontaneous efflux of both amino acids was rather slow but could be enhanced by their uptake inhibitors. Taurine efflux was facilitated by exogenous taurine, hypotaurine, -alanine and GABA, whereas GABA, nipecotic acid and homotaurine effectively enhanced GABA release. The stimulatory potency of the analogues closely corresponded to their ability to inhibit taurine and GABA uptake, respectively, indicating that these efflux processes could be mediated by the carriers operating outwards. Glutamate induced GABA release, whereas taurine efflux was potentiated by aspartate, glutamate, cysteate, homocysteate and kainate. The centrally acting drugs, including GABA agonists and antagonists, as well as the proposed taurine antagonist TAG (6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide), had no marked effects on spontaneous taurine and GABA release. Potassium ions stimulated dosedependently both taurine and GABA release from the slices, the responses of taurine being strikingly slow but sustained. Exogenous GABA and nipecotic acid accelerated the potassium-stimulated GABA release, whereas picrotoxin and bicuculline were ineffective. The potassium-stimulated taurine release was unaltered or suppressed by exogenous taurine and analogues, differing in this respect from GABA release. The apparent magnitude of the depolarization-induced GABA release is thus influenced by the function of membrane transport sites, but the same conclusion cannot be drawn with regard to taurine. Haloperidol and imipramine were able to affect the evoked release of both taurine and GABA.     

Rapid and sensitive step gradient assays of glutamate, glycine, taurine and γ-aminobutyric acid by high-performance liquid chromatography–fluorescence detection with o-phthalaldehyde–mercaptoethanol derivatization with an emphasis on microdialysis samples

We developed a rapid step-gradient HPLC method for determination of glutamate, glycine and taurine, and a separate method for determination of γ-aminobutyric acid (GABA) in striatal microdialysates. The amino acids were pre-column derivatized with o-phthalaldehyde–2-mercaptoethanol by using an automated refrigerated autoinjector. Separation of the amino acids was established with a non-porous ODS-II HPLC column, late-eluting substances were washed out with a one-step low-pressure gradient. Concentrations of the amino acids were determined with a fixed-wavelength fluorescence detector. The detection limit for GABA was 80 fmol in a 15 μl sample, detection limits for glutamate, glycine and taurine were not determined because their concentrations in striatal perfusates were far above their detection limits. Total analysis time was less than 12 min, including the wash-out step. The methods described are relatively simple, sensitive, inexpensive, and fast enough to keep up with the microdialysis sampling.     

GABA and glycine in synaptic vesicles: storage and transport characteristics.

gamma-Aminobutyric acid (GABA) and glycine are major inhibitory neurotransmitters that are released from nerve terminals by exocytosis via synaptic vesicles. Here we report that synaptic vesicles immunoisolated from rat cerebral cortex contain high amounts of GABA in addition to glutamate. Synaptic vesicles from the rat medulla oblongata also contain glycine and exhibit a higher GABA and a lower glutamate concentration than cortical vesicles. No other amino acids were detected. In addition, the uptake activities of synaptic vesicles for GABA and glycine were compared. Both were very similar with respect to substrate affinity and specificity, bioenergetic properties, and regional distribution. We conclude that GABA, glycine, and glutamate are the only major amino acid neurotransmitters stored in synaptic vesicles and that GABA and glycine are transported by similar, if not identical, transporters.     

Optimal reaction conditions for the production of γ-aminobutyric acid by the marine yeast isolate Pichia anomala MR-1 strain

The optimal conditions for the production of γ-aminobutyric acid (GABA) by the marine yeast isolate Pichia anomala MR-1 strain appeared to be completely different from those required for growth and biomass production of the yeast strain. By proper reaction temperatures and pH levels, the efficiency of GABA production was improved dramatically. Supplying enough energy for the reaction was another important technical element to improve the production efficiency of GABA. The MR-1 isolate was found to use various saccharides, including glucose and fructose, as well as intermediate metabolites in the tricarboxylic acid (TCA) cycle to promote GABA production. On the other hand, ethanol, various free amino acids, and organic acids were detected along with GABA in the reaction solution. These results suggest that production of GABA by the MR-1 strain is not due to a single enzymatic reaction. Rather it is a fermentation reaction, possibly involving a combination of intracellular metabolic pathways.     

ON THE SITE OF ORIGIN OF TRANSMITTER AMINO ACIDS RELEASED BY DEPOLARIZATION OF NERVE TERMINALS IN VITRO

Abstract— —The site of origin of transmitter amino acids released by depolarizing agents from nerve endings was studied. The model used was the incubated and depolarized synaptosome preparation from which the component soluble, synaptic vesicle, membrane and mitochondrial sub-fractions were obtained. Synaptosomal amino acids were radioactively labelled from D-[U-¹⁴C]glucose in vivo by intraventricular injection and in vitro during subsequent incubation. The specific radioactivities of amino acids released in response to K⁺ (56 mM) or veratrine (75 μM) were found to closely resemble those of the soluble cytoplasmic fraction, in most cases differing significantly from those of the other fractions. The specific radioactivity of the GABA and aspartate released by K⁺ stimulation and the GABA and glutamate released by veratrine were significantly different from that of the vesicles in each case. The specific radioactivities of glutamate released by both agents, and also GABA with K⁺ stimulation, were approximately double that of the amino acid released in control conditions. Depletion of the soluble cytoplasmic pools of glutamate, GABA and aspartate occurred following stimulation, corresponding to the induced-release of these compounds. Turnover of the amino acids in the other subfractions was too low to account for their participation in the release process in addition to the soluble cytoplasmic pool. A cytoplasmic origin of release of neurotransmitter amino acids from nerve endings is proposed.     

The effects of two anticonvulsants on amino acid levels in the developing rat cerebellum

Two anticonvulsants were administered pre- and postnatally to determine their effects on putative amino acid neurotransmitter levels in the rat cerebellum. The amino acids were quantitated using precolumn fluorescence derivatization and reverse-phase high performance liquid chromatography at various postnatal intervals. Treatment with clonazepam produced an initial depression in levels of most of the amino acids analyised. By three weeks postnatal all the amino acids, with the exception of GABA, had returned to control levels. GABA levels were still depressed five weeks after the cessation of treatment. Phenobarbital treatment produced an initial elevation in the level of GABA. At three weeks postnatal, both GABA and glutamate levels were elevated and remained so at eight weeks postnatal. In conclusion, the data demonstrated that each anticonvulsant produced unique, acute and chronic alterations in the levels of the cerebellar amino acids.     

Effect of exogenous amino acids,glucose and citric acid on the patterns of short-term accumulation and loss of amino acids in the root-zone of sand-cultured forage rape (Brassica napus L.)

Amino acid loss from the roots of 25-day-old, sterile and non-sterile sand-grown forage rape plants, was determined over periods of up to 3.5 hours. Amino acid accumulation in the root-zone of sterile plants was concentration-dependent giving a convex accumulation profile. Amino acid levels in the root zone of non-sterile plants rapidly attained steady state values. Microbial assimilation of amino acids within the root zone appeared to lower amino acid concentrations, resulting in an underestimation of rates of amino acid loss from roots. The concentrations of most amino acids were higher after selected amino acids were supplied to the root zone. The response to exogenous acids was dependent on the concentration and composition of the acids added. Addition of a mixture containing ASN, GLN and GABA, each at 0.25 mM resulted in a greater increase in individual and total acid levels compared with a mixture containing ALA, SER, GLY and THR at the same concentration. Apparently, amino acids supplied exogenously competed with acids lost from the plant, by providing an alternative nutrient source for root zone micro-organisms. Addition of glucose and citric acid had a similar effect to addition of ALA, SER, GLY and THR, but were less effective than ASN, GLN and GABA at all concentrations tested. The nitrogen-rich amino acids ASN and GLN, and the -amino acid, GABA, appeared to compete more effectively with plant-derived acids than did ALA, SER, GLY and THR, the most abundent constituents of the plant-derived acids, which had the highest calculated rates of microbial consumption. Therefore, although bacterial consumption showed a dependence on amino acid concentration, a degree of selectivity for nitrogen-rich acids and gaba was also apparent.     

The stimulus-evoked release of glutamate and GABA from brain subregions following transient forebrain ischemia in the rat

The release of glutamate and GABA in response to K⁺ depolarization was determined for tissue prisms prepared from brain subregions removed from rats following 30 min of forebrain ischemia or recirculation periods up to 24 h. There were statistically significant effects of this treatment on release of both amino acids from samples of the dorsolateral striatum, an area developing selective neuronal degeneration. However, for at least the first 3 h of recirculation the calcium-dependent and calcium-independent release of both amino acids in this region were similar to pre-ischemic values. Differences were observed under some conditions at longer recirculation times. In particular there was a decrease in calcium-dependent GABA release at 24 h of recirculation and a trend towards increased release of glutamate at 6 h of recirculation and beyond. No statistically significant differences were seen in samples from the paramedian neocortex, a region resistant to post-ischemic damage. These results suggest that changes in the ability to release glutamate and GABA in response to stimulation are not necessary for the development of neurodegeneration in the striatum but rather that release of these amino acids may be modified as a result of the degenerative process.     

EFFECTS OF TETRODOTOXIN, CALCIUM AND MAGNESIUM ON THE RELEASE OF AMINO ACIDS FROM SLICES OF GUINEA-PIG CEREBRAL CORTEX

Abstract— Tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ were used in an effort to identify the portion of the evoked release of endogenous amino acids, labelled via metabolism of [¹⁴C]-glucose, and several exogenous labelled amino acids, that came from nerve terminals when slices of guinea pig cerebral cortex were superfused with glucose-free solutions and stimulated electrically. With some exceptions, spontaneous release of labelled amino acids was decreased by 2 μm -tetrodotoxin but increased in Ca²⁺-free medium and in solutions containing an extra 24 mm -MgCl₂. Tetrodotoxin suppressed 85–90% of the stimulated release of almost all labelled amino acids, but had a smaller effect on the release of endogenous ¹⁴C-labelled threonine-serine-glutamine (unseparated). In Ca²⁺-free solution, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 80–90%, but that of endogenous ¹⁴C-labelled threonine-serine-glutamine was unaffected as was most of the release of the other labelled amino acids. In medium containing an extra 24mM-MgCl₂, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 75-85%, that of exogenous labelled aspartate and GABA by 50–65%, but the release of the other labelled amino acids was unaffected. The control stimulated releases of endogenous ¹⁴C-labelled glutamate, aspartate and GABA were much larger than those of other labelled amino acids but were reduced by tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ to a level similar to that of the control stimulated releases of the other labelled amino acids. These results suggest that almost all of the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA came from nerve terminals while those of the other labelled amino acids came from other tissue elements. In addition, they are in accord with a transmitter role for glutamate, aspartate and GABA in cerebral cortex.     

Changes with aging in the levels of amino acids in rat CNS structural elements I. Glutamate and related amino acids

Glutamate and related amino acids were determined in 53 discrete brain areas of 3-and 29-month-old male Fischer 344 rats microdissected with the punch technique. The levels of amino acids showed high regional variation-the ratio of the highest to lowest level was 9 for aspartate, 5 for glutamate, 6 for glutamine, and 21 for GABA. Several areas were found to have all four amino acids at very high or at very low level, but also some areas had some amino acids at high, others at low level. With age, in more than half of the areas, significant changes could be observed, decrease occurred 5 times more frequently than increase. Changes occurred more often in levels of aspartate and GABA than in those of glutamate or glutamine. The regional levels of glutamate and its related amino acids show severalfold variations, with the levels tending to decrease in the aged brain.     

Photochemical Damage in the Albino Rat Retina: Morphological Changes and Endogenous Amino Acids

Abstract: Endogenous amino acids were measured in retinas of rats exposed for up to 48 h to fluorescent light. Typical light damage was seen in photo–receptor cells after 30 h exposure to a maximum luminance of 1544 scotopic lux; and, from this time, taurine levels were significantly reduced. In contrast, the concentrations of other amino acids increased. After 18 h exposure to light, GABA, glycine, glutamate, and aspartate levels were raised in the photo-receptor cells, and GABA, glutamate, and glutamine levels in the inner retina. When ‘exposed’ animals were returned to their normal environment for 72 h, photoreceptor degeneration progressed and taurine concentrations were further reduced: the results suggest that the loss was from damaged photo–receptor cells. At this time the concentrations of the other amino acids measured had, in general, returned to normal