Excitatory sulphur amino acid-evoked neurotransmitter release from rat brain synaptosome fractions

Summary The neuroactive sulphur-containing amino acids L-cysteate (CA), L-cysteine sulphinate (CSA), L-homocysteine sulphinate (HSA), S-sulpho-L-cysteine (SC) and L-homocysteate (HCA) evoked the release of previously accumulated D-[³H]aspartate from rat brain cerebrocortical and cerebellar synaptosome fractions in a manner that was wholly Ca²⁺-independent. However, analysis of endogenous release by hplc revealed the presence of both Ca²⁺-dependent and -independent components of L-glutamate release but only a Ca²⁺-independent component of L-aspartate release. CA, CSA, HSA and SC but not HCA evoked the release of previously accumulated [³H]GABA from synaptosome fractions by a mechanism shown to comprise both a Ca²⁺-dependent and -independent component. The specific antagonists of the N-methyl-D-aspartate (NMDA) receptor, 3-[(±)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (CPP) and the relatively selective competitive quisqualate (QUIS)/kainate (KA) receptor antagonist, 6-cyano-7-dinitroquinoxalinedione (CNQX), were ineffective in blocking the excitatory sulphur amino acid-evoked release of either D-[³H]aspartate, [³H]GABA or of endogenous established transmitter amino acids.     

Studies on synaptic vesicles in mammalian brain characterization of highly purified synaptic vesicles from bovine cerebral cortex

Synaptic vesicles have been isolated from bovine cerebral cortex by sequential differential and density gradient centrifugations followed by chromatography on a Sepharose 6B column. We have studied the morphology, enzymatic markers, neurotransmitter and ATP contents and protein composition of the vesicles. The specific contents of acetylcholine, gamma-aminobutyric acid, aspartate, glutamate and catecholamines were 4--8-fold higher in the vesicle fraction compared to the crude synaptosomal pellet. Electron micrographs of the vesicle preparation showed enrichment of vesicular material with an average diameter of 50 nm. The purity of the preparation was assessed by the very low activities of enzymatic markers of cellular membranes and cytosol components. Some Ca--Mg-activated ATPase activity was detected in the vesicle preparations, but its content relative to the neurotransmitters fell on chromatography, suggesting that this activity may be partially contributed by non-synaptic vesicle components, such as small microsomes. The isolated synaptic vesicles were solubilized with 1% sodium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis. The major Coomassie blue stained bands observed with apparent molecular weights of 160,000 and 55,000 were enriched in parallel to the increase in purity of the preparation.     

Neurotransmitter amino acids in the CNS. II. Some changes in amino acid levels in rat brain synaptosomes during and after in vitro anoxia and simulated ischemia

The effects of in vitro anoxia and membrane depolarization by veratridine on the uptake and release of amino acids were investigated in suspensions of synaptosomes isolated from the forebrains of rats. It was observed that GABA, aspartate and glutamate were released from synaptosomes in anaerobic conditions and upon addition of veratridine in a time-dependent manner. The release of the two latter amino acids was faster and more pronounced than that of GABA. The other amino acids were not affected in any systematic way by either condition. Re-introduction of oxygen or addition of tetrodotoxin to veratridine-treated synaptosomes resulted in the re-uptake of GABA, aspartate and glutamate, which was much faster and more complete for GABA than for the acidic amino acids, especially at acid pH values. The amounts of aspartate and glutamate in the incubation mixture remained constant during all the manipulations whereas that of GABA increased by about 30% during anaerobiosis, in agreement with the results obtained during in vivo ischemia. It is postulated that synaptosomes which utilized glutamate and aspartate as neurotransmitters are more damaged by anoxia and depolarization with veratridine than the population which utilizes GABA. These observations may explain reports that those neurons which are thought to receive major glutamatergic input are particularly sensitive to the lack of oxygen.     

Neurotransmitter amino acids in the CNS. I. Regional changes in amino acid levels in rat brain during ischemia and reperfusion

The levels of amino acids in 6 regions of the brain (cortex, hippocampus, striatum, diencephalon, stem and cerebellum) were determined during an ischemic insult of 30 min and after recovery periods of up to 10 h. The results were analyzed in two groups: putative neurotransmitters (GABA, aspartate, glutamate, taurine, glycine and alanine) and non-neurotransmitters. In the neurotransmitter group, it was found that at the end of 30 min ischemia the levels of aspartate and glutamate slightly decreased whereas those of GABA and alanine rose substantially. The amounts of glycine and taurine remained unchanged. In 30 min after the ischemic insult, there were much larger decreases in aspartate and glutamate and increases in GABA and alanine with no change in glycine and taurine. At 2 h recovery the levels of the neurotransmitter amino acids had almost returned to control values and were fully recovered by 10 h after ischemia. It is postulated that glutamate and aspartate are released during ischemia into the extracellular space and subsequently ‘washed-out’ into the blood during the reperfusion. Release of GABA, if it occurs, is however, compensated by increase in its synthesis and decrease in its degradation under anaerobic conditions, both of which contribute to the rise in its steady-state level. In the non-transmitter category, increases were seen in amino acids present normally in very small concentrations; tyrosine, lysine, leucine and 3 hydrophobic amino acids: valine, methionine and phenylalanine, which were most pronounced at 2 h after ischemia. It is suggested that the rise in the levels of these molecules is the consequence of stimulation of protein breakdown caused by activation of intracellular proteases by calcium and H⁺ during the ischemic episode. Regional variations in the patterns of changes were small although in the ischemic model used the brainstem seemed to be least affected.     

Impact of STZ‐induced hyperglycemia and insulin‐induced hypoglycemia in plasma amino acids and cortical synaptosomal neurotransmitters

In this work, we evaluated the effects of streptozotocin (STZ)‐induced hyperglycemia and an acute episode of insulin‐induced hypoglycemia in plasma amino acids and cortical neurotransmitters. For that purpose, we used citrate (vehicle)‐treated Wistar rats, STZ‐treated rats [i.p., 50 mg/kg body weight], and STZ‐treated rats injected with insulin [s.c., dose adjusted with blood glucose levels] 1 h prior to sacrifice to induce an acute episode of hypoglycemia. Plasma was collected for determination of amino acids levels. In addition, cortical synaptosomal preparations were obtained and the total levels of neurotransmitters, levels of aspartate, glutamate, taurine, and GABA released by the action of KCl, iodoacetic acid (IAA), ouabain, and veratridine, membrane potential and ATP levels were evaluated. Compared with control rats, plasma from hypoglycemic rats presented increased levels of aspartate, glutamate, glutamine, and taurine whereas GABA levels were decreased in STZ and hypoglycemic rats. Similarly, glutamate and taurine levels were increased in hypoglycemic synaptosomes while GABA decreased in hypoglycemic and STZ‐diabetic synaptosomes. The depolarizing agent KCl promoted an increase in aspartate, glutamate, and taurine release from hypoglycemic synaptosomes. The highest release of neurotransmitters occurred in the presence of veratridine and ouabain, two other depolarizing agents, in all groups of experimental animals. However, a higher release of glutamate was observed in the diabetic and hypoglycemic synaptosomes. No alterations were observed in synaptosomal membrane potential and ATP levels. These results show that in the presence of a metabolic insult a higher release of excitatory amino acids occurs, which may underlay the neuronal injury observed in type 1 diabetic patients under insulin therapy. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

Effects of ageing on the content in sulfur-containing amino acids in rat brain

Summary Concentrations of the sulfur-containing amino acids methionine, homocysteic acid, cysteic acid and taurine were measured in brain structures of young and old Wistar rats in an attempt to etablish a possible link between the increase in oxidative stress with ageing and changes in tissue levels of these amino acids. Contrary to data reported by others, in all brain structures of young and old rats homocysteic acid levels could not be quantified. Compared with young rats, in old animals taurine and methionine concentrations significantly decreased in striatum and cortex; decreased taurine levels were also found in nucleus accumbens and cerebellum and lower concentrations of methionine were found in midbrain, hippocampus and pons-medulla. Cysteic acid levels either did not change or significantly increased in cortex and hippocampus. These results are discussed taking into account the biosynthesis of sulfur-containing amino acids in rat brain and the decrease in glutathione in relation to oxidative stress with ageing.Changes in aspartic acid, glutamic acid, serine, glutamine, glycine and GABA concentrations with ageing were also determined in the same brain structures and were in good agreement with those previously reportedAbbreviations H ₂O₂ hydrogen peroxide - MAO monoamine oxidase - GSHP glutathione peroxidase - PAPS 3-phosphoadenosine-5-phosphosulfate - OPA O-phthaldialdehyde - HPLC high performance liquid chromatography - Asp aspartic acid - CA cysteic acid - CSA cysteine sulfinic acid - Cys cysteine - GABA -aminobutyric acid - Gln glutamine - Glu glutamic acid - Gly glycine - HCA homocysteic acid - Met methionine - Ser serine - Tau taurine     

Transsynaptic modulation of the synaptic vesicle cycle by cell-adhesion molecules

Delicate control of the synaptic vesicle cycle is required to meet the demands imposed on synaptic transmission by the brain's complex information processing. In addition to intensively analyzed intrinsic regulation, extrinsic modulation of the vesicle cycle by the postsynaptic target neuron has become evident. Recent studies have demonstrated that several families of synaptic cell-adhesion molecules play a significant role in transsynaptic retrograde signaling. Different adhesion systems appear to specifically target distinct steps of the synaptic vesicle cycle. Signaling via classical cadherins regulates the recruitment of synaptic vesicles to the active zone. The neurexin/neuroligin system has been shown to modulate presynaptic release probability. In addition, reverse signaling via the EphB/ephrinB system plays an important role in the activity-dependent induction of long-term potentiation of presynaptic transmitter release. Moreover, the first hints of involvement of cell-adhesion molecules in vesicle endocytosis have been published. A general hypothesis is that specific adhesion systems might use different but parallel transsynaptic signaling pathways able to selectively modulate each step of the synaptic vesicle cycle in a tightly coordinated manner.     

Effects of excitatory amino acids,and of their agonists and antagonists on the release of neurotransmitters from the chick retina

Effects of glutamic, aspartic, and cysteic acid, and of kainic acid and N-methyl aspartate on the release of labeled GABA, glycine, and taurine were examined in isolated, perfused chick retina. Glutamic acid (0.5-2 mM), increased the release of ³H-GABA by more than four times and that of ¹⁴C-glycine by about two times. The release of GABA decreased 50% and that of glycine 95% in the presence of the antagonist of glutamic acid receptors, glutamate diethyl ester (300 m?M). N-methyl aspartate, used as an agonist of aspartic acid receptors, preferentially increased the release of GABA (seven times) over that of glycine (three times). The stimulatory effect of N-methyl aspartate was antagonized by D-α aminoadipate and by Mg. Kainic acid (10 m?M) induced the release of glycine but not that of GABA. Cysteic acid failed to modify the release of any of the amino acids examined. The efflux of labeled taurine was practically unaffected by all the compounds utilized. The release of GABA by the excitatory amino acids and agonists was Ca-independent but Na-dependent, whereas the release of glycine was markedly Ca-dependent. The evidence presented here suggests that experimental conditions activating receptors of excitatory amino acids differently affect the release of inhibitory amino acids.     

The effects of chlordiazepoxide on synaptic transmission and amino acid neurotransmitter release in slices of rat olfactory cortex

The rat olfactory cortex slice has been used to investigate the effects of chlordiazepoxide on evoked field potentials and the release of endognous amino acid neurotransmitters (aspartate, glutamate, GABA and possibly taurine) which accompany electrical stimulation of the lateral olfactory tract. When single, low frequency stimuli were employed, chlordiazepoxide (2 μM-1 mM) depressed the amplitude of the field potential correlate of the depolarizing actions of the lateral olfactory tract excitatory transmitter (aspartate?) although aspartate release was unaffected. The field potential correlate of GABA-mediated presynaptic inhibition (late N-wave) was also depressed in amplitude but low durg concentrations (between approximately 2 and 50 μM) increased its peak duration. Effects of chlordiazepoxide on evoked inhibition were analyzed by giving paired stimuli such that the second stimulus occured during the field potentials evoked by the first stimulus. Chlordiazepoxide (1–20 μM) increased the depression in amplitudes of the presynaptic massed action potential and late N-wave evoked by the second of a pair of stimuli compared with those evoked by the first stimulus suggesting that presynaptic inhibition was potentiated. These effects of chlordiazepoxide were accompanied by a significant reduction in aspartate release from the lateral olfactory tract terminals. Moreover, the drug effects on presynaptic inhibition and aspartate release were antagonized by picrotoxin (5 μM). On the other hand, chlordiazepoxide (1–50 μM) had no significant effect on postsynaptic inhibition. The results are discussed in terms of both the sites (presynaptic or postsynaptic) and mechanisms of action of chlordiazepoxide.     

Synaptosomes prepared from fresh human cerebral cortex; morphology, respiration and release of transmitter amino acids

Synaptosomes prepared from fresh human cerebral cortex were shown to be morphologically similar to those from other species. On incubation, they took up oxygen at a high and linear rate and accumulated potassium against a concentration gradient. In response to depolarization by raised extracellular K+ or addition of veratrine, they showed increased respiration, lowered tissue potassium, and enhanced release of glutamate, aspartate and GABA. The preparation may be of value for studies of neurological disorders.     

Antagonists of synaptic and amino acid excitation of neurones in the cat spinal cord

In the spinal cord of the anaesthetized cat microelectrophoretically administered (+/-)-cis-2,3-piperidine dicarboxylate (2,3-PDA), (+/-)-cis-2,5-piperidine dicarboxylate (2,5-PDA), gamma-D-glutamylglycine (gamma DGG), beta-D-aspartyl-beta-alanine (beta DAA), (+/-)-2-amino-4-phosphonobutyrate (2-APB), (+/-)-2-amino-5-phosphonovalerate (2-APV) and (+/-)-2-amino-7-phosphonoheptanoate (2-APH) were assessed as antagonists of chemical excitation of dorsal horn interneurones and Renshaw cells by N-methyl-D-aspartate (NMDA), L-aspartate, quisqualate (QUIS), kainate and L-glutamate, and of monosynaptic and polysynaptic excitation by impulses in primary afferent fibres of muscle and cutaneous origin. Whereas polysynaptic excitation of interneurones was readily and reversibly depressed by 2-APV, 2-APH, beta DAA, gamma DGG and 2,3-PDA, all of which also reduced excitation by NMDA (and L-aspartate) more than that by QUIS (and L-glutamate), no selective antagonism of monosynaptic excitation could be demonstrated. In particular, 2,3-PDA, which depressed excitation by kainate to a greater extent than that by either QUIS or NMDA, appeared to have no effect on monosynaptic excitation. The results support the involvement of L-aspartate as the transmitter of some spinal excitatory interneurones, but none of the antagonists tested were considered suitable for assessing the role of L-glutamate as the transmitter of some spinal primary afferent fibres.     

Differential effects of hypertonic mannitol and glycerol on rat brain metabolism and amino acids

Intraperitoneal injections in rats of two different dosages of hypertonic solutions containing mannitol or glycerol caused complex and differential changes in brain amino acids. When plasma osmolalities were elevated to toxic levels of 397–432 mOsm/kg H₂O, brain sodium was increased, whereas plasma sodium was decreased. Brain potassium was not affected. Brain water decreased significantly, concomitant with elevation of plasma osmolality. Both brain lactic acid and [¹²⁵I]albumin space rose significantly. Brain amino acids (mostly aliphatic and basic amino acids) as well as GABA and glycine (putative inhibitory neurotransmitters) increased after both mannitol and glycerol. Ammonia was stimulated by mannitol but was unaffected by glycerol. Plasma amino acids, which generally increased after mannitol, were decreased by glycerol. When the plasma osmolalities were elevated only to moderate levels (about 350 mOsm/kg H₂O, only glycerol induced a significant increase in brain taurine, aspartic acid, alanine, leucine and lysine. Thus, with moderate hyperosmolality, glycerol has striking effects on brain amino acid metabolism that are not observed with mannitol.     

Metabolism of amino acids in the brain

In this article, we reviewed the specificity of amino acid metabolism in the brain. We considered the results of many basic studies that supported a role of amino acids as neurotransmitters, substrates for synthesis of neuroactive peptides, proteins, and other biologically active substances in the CNS. These data suggest that changes in the pool of amino acids may be involved in the development of CNS pathologies.     

Membrane protein and glycoprotein composition of beef brain synaptic vesicles

Synaptic vesicles were isolated from adult bovine cortical gray matter by differential centrifugation and membrane filtration of a hypoosmotically lysed crude mitochondrial fraction. Vesicle preparations were analyzed for purity by electron microscopy and enzyme assays. Polyacrylamide gel electrophoresis of SDS-solubilized and 2-mercaptoethanol-reduced vesicle membrane proteins revealed 4 major proteins with molecular weights ranging from 17,000 to 60,000, and about 10 minor proteins with molecular weights up to 170,000. The protein profile of the Triton X-100-extracted vesicle membranes was less complex, with 1 major protein and 5 minor bands. The major protein of the Triton extract was identified as a glycoprotein with a molecular weight of 45,000. Two additional minor PAS-positive bands were seen, with molecular weights of 78,000 and 95,000.     

Autoradiographic mapping of synaptic vesicle glycoprotein 2A in non-human primate and human brain

Synaptic vesicle glycoprotein 2A (SV2A) has been previously characterized as an imaging biomarker for assessment of synaptic density in positron emission tomography (PET) studies of patients with neurological conditions. To provide detailed maps of the brain localization of SV2A autoradiography studies were carried out using the SV2A radioligand [¹¹C]UCB-J and whole hemisphere sections of non-human primate (NHP) and human brain. Binding of [¹¹C]UCB-J was observed in all evaluated grey matter structures of the primate brain, with highest density in the caudate nucleus and cortex and lowest density in pons and globus pallidus. The density of [¹¹C]UCB-J binding sites in human brain showed a good correlation with that in NHP brain. Binding of [¹¹C]UCB-J in the white matter was very low relative to that in grey matter containing structures and was only inhibited to a minor extent by co-incubation with a saturating concentration of unlabelled UCB-J. The high-resolution images obtained in the present study may aid the interpretation of data acquired in human subjects examined using [¹¹C]UCB-J in PET studies. In addition, observation of low binding for [¹¹C]UCB-J in white matter (centrum semiovale) supports that this structure can be used as a reference region for quantitative analysis of [¹¹C]UCB-J PET data.     

Electrochemical detection of human brain transmitter amino acids by high-performance liquid chromatography of stable O-phthalaldehyde-sulphite derivatives

Summary A simple, sensitive, reliable and reproducible isocratic HPLC technique for the measurement of OPA/sulphite derivatives of human brain amino acid neurotransmitters is described. This employs a sample preparation that is also compatible with the concurrent determination of monoamines and their metabolites on a separate HPLC system. The method has been applied to the determination of GABA and glutamate in brain tissue taken post-mortem from patients with Huntington's disease and control subjects.This work was supported by the Wellcome Trust and the Association to Combat Huntington's Chorea. Brain tissue was provided by the Cambridge Brain Bank Laboratory. We thank Dr. M. Joseph for drawing our attention to the potential of this technique.     

Cerebral uptake of amino acids in human subjects and rhesus monkeys in vivo

In experiments with 11 human subjects and 12 rhesus monkeys given ¹⁴C-labeled amino acids intravenously, blood samples were drawn simultaneously from the femoral artery (A) and the superior bulb of the internal jugular vein (V). Analyses of labeled and unlabeled free amino acids indicated that both A-V and V-A differences resulted with each amino acid. In no case were there consistent A-V or V-A differences (excepting with L-[methyl-¹⁴C] methionine where consistent A-V differences resulted). However, in experiments with rhesus monkeys, from 0.2% to 2.3% of the injected ^14C-labeled amino acids was taken up by brains 90–120 minutes after injection. Therefor, it was suggested that, in humans and monkeys in vivo, small but significant amounts of amino acids enter the brain from blood over a period of time by a bidirectional exchange mechanism.     

Free amino acid pool in the brain of mice homozygous for the gene "dilute lethal"

The neurologic mutant "dilute lethal" (dl) mice, which reveal several neurologic and biochemical disturbances similar to human phenylketonuria, were used to investigate some aspects of amino acid disorder. We have studied the free amino pool in the brain of "dl" mice and of their control littermates as well as phenylalanine and tyrosine levels in brain and liver as a function of age and after phenylalamine overload. The tyrosine level decreased in brain and liver of affected mice whereas the phenylalanine/tyrosine ratio increased as a function of age. The significantly higher phenylalanine level and phenylalanine/tyrosine ratio in the liver of 20-day-old "dl" mice suggest a lower liver phenylalanine hydroxylase activity. After phenylalanine overload, the impairment of phenylalanine metabolism is predominant in the brain of "dl" mice, suggesting a disturbance in phenylalanine hydroxylation. A decrease in the level of several amino acids occurs in the brains of "dl" mice without or after phenylalanine overload; these facts might correspond to a disturbance in the transfer of amino acids to the brain and may lead to impairment in protein synthesis.     

Tissue-type plasminogen activator triggers the synaptic vesicle cycle in cerebral cortical neurons

The active zone (AZ) is a thickening of the presynaptic membrane where exocytosis takes place. Chemical synapses contain neurotransmitter-loaded synaptic vesicles (SVs) that at rest are tethered away from the synaptic release site, but after the presynaptic inflow of Ca⁺² elicited by an action potential translocate to the AZ to release their neurotransmitter load. We report that tissue-type plasminogen activator (tPA) is stored outside the AZ of cerebral cortical neurons, either intermixed with small clear-core vesicles or in direct contact with the presynaptic membrane. We found that cerebral ischemia-induced release of neuronal tPA, or treatment with recombinant tPA, recruits the cytoskeletal protein βII-spectrin to the AZ and promotes the binding of SVs to βII-spectrin, enlarging the population of SVs in proximity to the synaptic release site. This effect does not require the generation of plasmin and is followed by the recruitment of voltage gated calcium channels (VGCC) to the presynaptic terminal that leads to Ca⁺²-dependent synapsin I phosphorylation, freeing SVs to translocate to the AZ to deliver their neurotransmitter load. Our studies indicate that tPA activates the SV cycle and induces the structural and functional changes in the synapse that are required for successful neurotransmission.