Regional distribution of putative amino acid neurotransmitters in the CNS of spiders (Arachnida: Araneida)

The regional occurrence of five amino acid neurotransmitters (GABA, taurine, glycine, glutamate, aspartate) was studied in the CNS of five spider families, using a fluorescence microchromatogram method. Clear differences in transmitter distribution were obvious in relation to the phylogenetic standard of the spider families and the CNS regions investigated. For example, high relative amounts of taurine and GABA were typically confined to the protocerebral brain parts, particularly in the Araneidae and Salticidae. Remarkable amounts of glutamate were shown to exist in the lower CNS parts of the Agelenidae and, especially, the Theraphosidae.The results obtained confirm the dominant functional role of the protocerebrum in arthropods.     

Sulfur Amino Acid Metabolism in the Developing Rhesus Monkey Brain: Subcellular Studies of Taurine, Cysteinesulfinic Acid Decarboxylase, γ-Aminobutyric Acid, and Glutamic Acid Decarboxylase

Abstract: Taurine, cysteinesulfinic acid decarboxylase (CSAD), glutamate, γ-aminobutyric acid (GABA), and glutamic acid decarboxylase (GAD) were measured in subcellular fractions prepared from occipital lobe of fetal and neonatal rhesus monkeys. In addition, the distribution of [³⁵S]taurine in subcellular fractions was determined after administration to the fetus via the mother, to the neonate via administration to the mother prior to birth, and directly to the neonate at various times after birth. CSAD, glutamate, GABA, and GAD all were found to be low or unmeasurable in early fetal life and to increase during late fetal and early neonatal life to reach values found in the mother. Taurine was present in large amounts in early fetal life and decreased slowly during neonatal life, arriving at amounts found in the mother not until after 150 days of age. Significant amounts of taurine, CSAD, GABA, and GAD were associated with nerve ending components with some indication that the proportion of brain taurine found in these organelles increases during development. All subcellular pools of taurine were rapidly labeled by exogenously administered [³⁵S]taurine. The subcellular distribution of all the components measured was compatible with the neurotransmitter or putative neuro-transmitter functions of glutamate, GABA, and taurine. The large amount of these three amino acids exceeds that required for such function. The excess of glutamate and GABA may be used as a source of energy. The function of the excess of taurine is still not clear, although circumstantial evidence favors an important role in the development and maturation of the CNS.     

Free amino acid pool in the brain of inbred strains of mice with different learning aptitudes]

GABA, glycine, taurine, putative inhibitory transmitters or modulators of the CNS are at a significantly lower level in the brains of SEC and DBA strains of mice which offer higher performances in avoidance and learning tests than in the C57 strain which scores poorly.     

Synthesis and Characterization of N,N-Dichlorinated Amino Acids: Taurine,Homotaurine, GABA and L-Leucine

Epilepsy, trauma and other circumstances leading to hyperexcitable conditions in the CNS tend neurochemically to be associated with excessive stimulated release of glutamic acid and/or a failure of GABA modulated inhibition. Somewhat to a lesser extent, taurine and its homologue homotaurine, have also been shown to antagonize the excitatory actions of glutamic acid. Here we report the successful synthesis and isolation in pure form of N,N-dichlorinated GABA, taurine, homotaurine and leucine. These compounds are much more lipophilic than their parent compounds and may therefore more readily penetrate the blood-brain barrier systems into the neural tissue, where they can be easily dechlorinated. Very preliminary biological testing shows that this may indeed occur. The synthesis and purification methodology will likely also be applicable to a number of other amino acids as well as certain peptides or selected proteins.     

Free amino acid levels in undernourished developing rat brain

Free amino acid levels in the brains of young ones born to mothers fed a 20% protein diet ad libitum (well nourished), 7.5% protein diet ad libitum (protein restricted) and a 20% protein diet in restricted amounts (pair-fed) were investigated during brain development in the present study. The dietary protein was obtained from a cereal-legume mixture. Protein restricted animals showed increases in the levels of taurine, glycine and glutamic acid and decreases in the concentrations of methionine, leucine, isoleucine, and GABA. The pair-fed animal showed increases only in glutamic acid and glycine and a decrease only in the levels of GABA. The significance of these observations is discussed.     

Anticonvulsant effects of some inhibitory neurotransmitter amino acids

The anticonvulsive effects of GABA, taurine, and glycine were investigated on several chemically-induced and genetic seizure models. Intravenous injections of either GABA, taurine, or glycine provided protection against 3-mercaptopropionic acid (MPA)-induced convulsions in adult Swiss mice. GABA was partially effective against isonicotinic acid hydrazide and was without effect against bicuculline-induced convulsions bProlonged administration of glycine prevented MPA-induced convulsions but not electrically induced seizures or seizures induced by strychnine or metrazol.Intragastric glycine protected young audiogenic seizure-susceptible DBA/2 mice against all three phases of sound-induced convulsions (wild running, clonic and tonic seizure), but GABA and taurine provided little or no protection. With increase of glycine, the cerebral levels of glutamine and serine also increased, but that of glutamic acid decreased. The endogenous glutamic and glycine levels were slightly higher in the brains of the audiogenic seizure-susceptible DBA/2 mice than in that of the resistant BALB/Cy strain.     

Changes in content of neuroactive amino acids and acetylcholine in the rat hippocampus following transient forebrain ischemia.

Changes in content of selected neuroactive amino acids [glutamic acid, aspartic acid, glycine, gamma-aminobutyric acid (GABA) and taurine] and acetylcholine (ACh) in the rat hippocampus following transient forebrain ischemia were investigated using male Wistar rats. Rats were allowed to survive for 1 or 5 days following 10 or 20 min of 4-vessel occlusion, and killed by a focused microwave irradiation. A significant reduction in all neuroactive amino acids examined except GABA was noted in the hippocampus on the fifth day. One day after the 4-vessel occlusion for 10 min, no significant effect on the content of neuroactive amino acids in all brain areas was observed. gamma-Aminobutyric acid content in the hippocampus was only significantly reduced on the fifth day after the occlusion for 20 min. Similarly, a significant decrease in ACh content in the hippocampus was observed on the fifth day after the occlusion for 20 min. Considering the data that a significant loss of neuronal cells in the hippocampus (delayed neuronal death) was detected only 5 days after the 4-vessel occlusion, it can be said that the alterations in the hippocampus of neuroactive amino acids such as glutamic acid, aspartic acid, glycine and taurine are more sensitive than those in GABA and ACh against cerebral ischemia. A possible correlation of these changes of neuroactive amino acids in the occurrence of delayed neuronal death in the hippocampus is also suggested.     

Effects of dorsal root section and occlusion of dorsal spinal artery on the neurotransmitter candidates in rat spinal cord

In order to obtain further evidence of putative neurotransmitters in primary sensory neurons and interneurons in the dorsal spinal cord, we have studied the effects of unilateral section of dorsal roots and unilateral occlusion of the dorsal spinal artery on cholinergic enzyme activity and on selected amino acid levels in the spinal cord. One week after sectioning dorsal roots from caudal cervical (C₇) to cranial thoracic (T₂) levels, the specific activity of choline acetyltransferase (ChAT) was significantly decreased and acetylcholinesterase (AChE) showed a tendency to decrease in the dorsal quadrant on the operated side of the spinal cord. Dorsal root sectioning had little effect on the levels of free glutamic acid or other amino acids in the dorsal spinal cord. These results suggest that primary sensory neurons may include some cholinergic axons, and that levels of putative amino acid transmitters are not regulated by materials supplied by axonal transport from the dorsal root ganglia. By contrast, one week following unilateral occlusion of the dorsal spinal artery, the activities of ChAT and AChE were unchanged in the operated quadrant of the spinal cord, while decreases of Asp, Glu, and GABA, and an increase in Tau were detected. These findings are consistent with the proposals that such amino acids, but not ACh, may function as neurotransmitter candidates in interneurons of the dorsal spinal cord.Abbreviation used ACh acetylcholine - AChE acetylcholinesterase - Asp aspartic acid - ChAT choline acetyltransferase - GABA -aminobutyric acid - Glu glutamic acid - Gly glycine - SP substance P - Tau taurine     

Elevation of cerebral levels of nonessential amino acids in vivo by administration of large doses

Taurine, aspartic acid, glutamic acid, glycine, and GABA were administered either intragastrically or in liquid diets to mice and rats. This resulted in a great increase in the plasma concentration of the administered amino acid, with plasma levels remaining elevated for several days.The prolonged increase in plasma levels resulted in significant increases in brain levels. Under these experimental conditions, taurine, aspartic acid, and glutamic acid were increased 30–60%; glycine and GABA 100%. During these experiments, plasma levels of taurine, aspartate, and glutamate were below brain levels; those of glycine and GABA were above.The findings show that even slowly penetrating amino acid levels can be increased in brain after parenteral administration of large doses.     

High-Affinity Transport of γ-Aminobutyric Acid, Glycine, Taurine, L-Aspartic Acid, and L-Glutamic Acid in Synaptosomal (P2) Tissue: A Kinetic and Substrate Specificity Analysis

In a cortical P2 fraction, [14C]gamma-aminobutyric acid ([14C]GABA), [14C]glycine, [14C]taurine, and [14C]glutamic and [14C]aspartic acids are transported by four separate high-affinity transport systems with L-glutamic acid and L-aspartic acid transported by a common system. GABA transport in cortical synaptosomal tissue occurs by one high-affinity system, with no second, low-affinity, transport system detectable. Only one high-affinity system is observed for the transport of aspartic/glutamic acids; as with GABA transport, no low-affinity transport is detectable. In the uptake of taurine and glycine (cerebral cortex and pons-medulla-spinal cord) both high- and low-affinity transport processes could be detected. The high-affinity GABA and high-affinity taurine transport classes exhibit some overlap, with the GABA transport system being more specific and having a much higher Vmax value. High-affinity GABA transport exhibits no overlap with either the high-affinity glycine or the high-affinity aspartic/glutamic acid transport class, and in fact they demonstrate somewhat negative correlations in inhibition profiles. The inhibition profiles of high-affinity cortical glycine transport and those of high-affinity cortical taurine and aspartic/glutamic acid transport also show no significant positive relationship. The inhibition profiles of high-affinity glycine transport in the cerebral cortex and in the pons-medulla-spinal cord show a significant positive correlation with each other; however, high-affinity glycine uptake in the pons-medulla-spinal cord is more specific than that in the cerebral cortex. The inhibition profile of high-affinity taurine transport exhibits a nonsignificant negative correlation with that of the aspartic/glutamic acid transport class.     

The subcommissural organ of the frog Rana perezi is innervated by nerve fibres containing GABA

The innervation of the frog subcommissural organ was studied by light-microscopic and ultrastructural immunocytochemistry using antisera against serotonin, noradrenaline, dopamine, gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, different GABA receptor subunits and bovine Reissner's fibre material (AFRU). In the proximity of the organ, serotonin- and noradrenaline-containing fibres were rare whereas dopamine-immunoreactive fibres were more numerous. Many GABA- and glutamic acid decarboxylase-containing nerve fibres were found at the basal portion of the ependymal cells of the subcommissural organ. Under the electron microscope, these GABA-immunolabelled nerve endings appeared to establish axoglandular synapses with secretory ependymal cells of the subcommissural organ. In addition, the secretory ependymal cells expressed high amounts of the beta2-subunit of the GABA(A) receptor. Since GABA-immunoreactive neurons were present in the frog pineal organ proper and apparently contributed axons to the pineal tract, we suggest that at least part of the GABAergic fibres innervating the frog subcommissural organ could originate from the pineal organ.     

Amino Acid Neurotransmitters and Dopamine in Brain and Pituitary of the Goldfish: Involvement in the Regulation of Gonadotropin Secretion

An isocratic high-performance liquid chromatographic technique was developed to measure levels of gamma-aminobutyric acid (GABA), glutamate, and taurine in the brain and pituitary of goldfish. Accuracy of this procedure for quantification of these compounds was established by evaluating anesthetic and postmortem effects and by selectively manipulating GABA concentrations by intraperitoneal administration of the glutamic acid decarboxylase (GAD) inhibitor 3-mercaptopropionic acid or the GABA transaminase inhibitor gamma-vinyl GABA. The technique provided a simple, rapid, and reliable method for evaluating the concentrations of these amino acids without the use of complex gradient chromatographic systems. To investigate the relationship between neurotransmitter amino acids and the control of pituitary secretion of gonadotropin, the effects of injection of taurine, GABA, or monosodium glutamate on GABA, glutamate, taurine, and, in some instances, monoamine concentrations in the brain and pituitary were evaluated and related to serum gonadotropin levels. Injection of taurine caused an elevation in serum gonadotropin concentrations. In addition, injection of the taurine precursor hypotaurine but not the taurine catabolite isethionic acid elevated serum gonadotropin levels. Intracerebroventricular injection of either GABA or taurine also elevated serum gonadotropin concentrations. Pretreatment of recrudescent fish with alpha-methyl-p-tyrosine reduced pituitary dopamine concentrations and also potentiated the serum gonadotropin response to taurine. Injection of monosodium glutamate caused an increase of glutamate content in the pituitary at 24 h; this was followed by a decrease at 72 h after administration. Pituitary GABA, taurine, and dopamine concentrations underwent a transient depletion after monosodium glutamate administration, and this was associated with an elevation of serum gonadotropin content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of taurine, 3-aminopropanesulfonic acid, and GABA on the hindgut and heart of the cockroach, Leucophaea maderae.

Taurine, glycine, glutamate, and gamma-aminobutyric acid (GABA) were all present in concentrations of greater than 1% of the total free amino acid content in the brain, thoracic, and abdominal ganglia of Leucophaea maderae. Hemolymph, subesophageal ganglia, and hindgut had substantial amounts of glutamate and glycine, but less than 0.3% taurine or GABA. Taurine, 3-aminopropanesulfonic acid (3-APS), cysteine-sulfinic acid (CSA), and GABA each had myotropic activity on the isolated cockroach hindgut, with 3-APS having the most consistent effect (ED50 = 0.63 mM), while taurine and CSA activities were similar to that of GABA on the hindgut. Both taurine and 3-APS had anti-arrhythmic effects on semi-isolated heart preparations of L. maderae, while GABA was inhibitory and induced arrhythmia. Bicuculline was antagonistic to the effects of GABA, taurine, and 3-APS on the hindgut, and induced arrhythmia in heart preparations; this arrhythmia was reversible by taurine, but not by GABA or 3-APS.     

EFFECTS OF AMINO-OXYACETIC ACID, ETHANOLAMINE-O-SULPHATE AND GABA ON THE CONTENTS OF GABA AND VARIOUS AMINES IN BRAIN SLICES

—The effects of amino-oxyacetic acid, ethanolamine-O-sulphate and γ-aminobutyric acid (GABA) on the contents of GABA, noradrenaline, dopamine and serotonin (5-HT) in slices of rat hypothalamus and midbrain were studied in vitro using a simultaneous fluorimetric assay procedure. Following control incubations the levels of 5-HT were raised, while the levels of the other substances remained steady. Amino-oxyacetic acid caused a reduction in the contents of noradrenaline and 5-HT, but had no effect on either GABA or dopamine. Ethanolamine-O-sulphate both raised the GABA content and lowered the noradrenaline content of slices, while the levels of dopamine and 5-HT were not altered. The presence of GABA in the incubation medium produced complex changes in these levels, depending both on the dose of GABA used and the brain area studied. In the hypothalamus, 0·07 mm -GABA caused an elevation in 5-HT, a drop in noradrenaline, and no change in either GABA or dopamine. With 5 mm -GABA, the noradrenaline level was raised slightly above control values and the endogenous GABA level doubled, while 5-HT and dopamine levels were not different from controls. Similar changes in 5-HT and GABA contents were observed with midbrain slices, but noradrenaline and dopamine were not affected. The possible modes of action of amino-oxyacetic acid and ethanolamine-O-sulphate on the amino acid and amine systems in the brain are discussed.     

Developmental changes of gamma-aminobutyric acid (GABA) and putative amino acid neurotransmitters in the organotypic culture of newborn mouse cerebellum

The quantitative changes and metabolism of GABA and putative amino acid neurotransmitters during early developmental stages in the organotypic culture of newborn mouse cerebellum were examined by using the high-performance liquid chromatograph (HPLC) technique. D-[U-14C]Glucose was used as a precursor of amino acids. To analyze amino acid neurotransmitters, explants were incubated for 4 weeks under standard conditions. The amount of GABA linearly increased from 8.7 +/- 1.3 nmol/mg protein (2 days in vitro, 2 DIV) to 26.5 +/- 6.1 nmol/mg protein (15 DIV) and was saturated after that (24.0 +/- 3.6 nmol/mg protein at 30 DIV). During the period of GABA increase, the capability for GABA synthesis from [14C]glucose increased rapidly from 3.03 +/- 0.67 nCi/mg protein (2 DIV, 3 h incubation) to 9.32 +/- 1.34 nCi/mg protein (15 DIV, 3 h incubation). In the case of glutamic acid, a putative neurotransmitter of granule cell parallel fibers in the cerebellum, the amount in explants was nearly constant during incubation, in contrast with the fact that the amount in vivo gradually increased. However, the capability for glutamic acid synthesis from [14C]glucose increased from 10.80 +/- 3.01 nCi/mg protein (2 DIV, 1 h incubation) to 27.62 +/- 4.71 nCi/mg protein (22 DIV, 1 h incubation). In the case of taurine, found in abundance in fetal brain and supposed to play a specific role in the development and maturation of the central nervous system, the amount in explants decreased from 139.8 +/- 4.0 nmol/mg protein (2 DIV) to 54.0 +/- 0.8 nmol/mg protein (30 DIV).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Taurine on Neurotransmitter Release from Insect Synaptosomes

The effect of taurine on the release of [3H]acetylcholine ([3H]ACH) and [3H]gamma-aminobutyric acid ([3H]GABA) from preloaded locust synaptosomes has been studied. Veratridine (100 microM) and K+ (100 mM) both evoked [3H]ACh release and this was reduced in a concentration-dependent manner by taurine (5, 10, and 20 mM). In contrast to this, veratridine induced no observable release of [3H]GABA, and the response to K+ was slight. In the presence of taurine, however, a concentration-dependent enhancement of [3H]GABA release was observed. Since nipecotic acid (1 mM), an inhibitor of neuronal GABA uptake, also revealed [3H]GABA release induced by veratridine, it is suggested that both this effect and that of taurine are due to prevention of GABA reuptake. These results suggest that taurine may act as a neuromodulator in insects.     

Use of reversed-phase high-performance liquid chromatography for simultaneous determination of glutamine synthetase and glutamic acid decarboxylase in crude extracts

Glutamine and γ-aminobutyric acid (GABA), formed from glutamic acid in crude tissue extracts by glutamine synthetase and glutamic acid decarboxylase respectively, were separated by derivatization with dansyl chloride followed by reversed-phase high-performance liquid chromatography on an Altex Ultrasphere ODS-5 column. The mobile phase was a gradient of 100 mM potassium dihydrogen phosphate (pH 2.1) with 0–40% acetonitrile. The amounts of glutamine and GABA formed from glutamic acid were determined under different reaction conditions.     

GABA Synthesis in Astrocytes After Infection with Defective Herpes Simplex Virus Vectors Expressing Glutamic Acid Decarboxylase 65 or 67

Abstract: Defective herpes simplex virus (HSV) vectors containing glutamic acid decarboxylase (GAD) cDNAs, either GAD65 or GAD67, were used to examine GAD function and GABA synthesis in rat cortical astrocytes, CNS cells that do not endogenously synthesize GABA. GAD vector infection resulted in isoform-specific expression of GAD as determined by western blotting and immunohistochemistry. Astrocytes infected with a β-galactosidase vector or uninfected expressed no GAD and contained no detectable GABA. GABA was detected in glial fibrillary acid protein-expressing cells after GAD65 vector infection. Significant amounts of GABA, as determined by HPLC, were synthesized in cultures infected with either GAD vector. The levels of GABA in GAD67 vector-infected cells were almost twofold higher than in GAD65 vector-infected cells. Vector infection did not alter levels of other intracellular amino acids. GABA was tonically released from astrocytes infected with the GAD67 vector, but no increase in release could be detected after treatment of the cells with K⁺, veratridine, glutamate, or bradykinin. The ability to transduce astrocytes so that they express GAD and thereby increase GABA levels provides a potential strategy for the treatment of neurologic disorders associated with hyperexcitable or diminished inhibitory activity.     

γ-aminobutyric acid in the central nervous system of metamorphosing and mature Manduca sexta

We have begun to examine the factors controlling the accumulation of the neurotransmitter γ-aminobutyric acid (GABA) in the central nervous system (CNS) of the sphinx moth Manduca sexta. Analysis of soluble amino acids in CNS structures from mature moths outlines the regional distribution of GABA. Analysis of amino acids in the antennal lobes (the primary olfactory centres) of Manduca during metamorphosis reveals that GABA accumulates gradually and continuously through most of adult development until eclosion; within 18 hr after eclosion, levels of GABA abruptly increase 27–50%. The activity of the biosynthetic enzyme glutamic acid decarboxylase (EC 4.1.1.15), assayed in extracts of antennal lobes from developing moths, does not change after eclosion. Extracts of hemolymph from mature moths contain low levels of glutamate ( <0.2 mM) and higher levels of certain other amino acids such as serine, glutamine and proline. The concentration of proline in hemolymph increases up to 2-fold after eclosion. Glutamate, glutamine and proline are interconvertible in the CNS, and each can serve as precursor for GABA synthesis both in vivo and in vitro. The efficiency of the precursor role in vitro is similar for each amino acid, as estimated from the ratio of the specific radioactivities of GABA and glutamic acid in the ganglion derived from each precursor. Exogenous proline and glutamine can equilibrate rapidly with the ganglionic pools of the same amino acids while glutamic acid is relatively excluded. Taken together, the findings of this study show that proline and glutamine may contribute substantially to synthesis of GABA in the CNS of M. sexta.     

Release of Endogenous Amino Acids from the Striatum from Developing and Adult Mice in Ischemia

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K⁺-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K⁺ stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K⁺ stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.