Concentrations Of Amino Acid Neurotransmitters Research Articles

Interstitial concentrations of amino acids in the rat striatum during global forebrain ischemia and potassium-evoked spreading depression.

The early detection and appropriate treatment of brain ischemia is of paramount importance. The interstitial concentrations of neurotransmitter amino acids are often used as an index of neuronal injury. However, monitoring of non-neurotransmitter amino acids may be equally important. We have studied the behavior of 10 amino acids during K(+)-induced spreading depression (application of 70 mM KCl during 40 min) and global forebrain ischemia (two-vessel occlusion with hypotension during 20 min). The concentrations of glutamate, aspartate, taurine, GABA, glycine, and alanine, measured in the rat striatum by microdialysis, increased during both ischemia and spreading depression, whereas glutamine concentrations decreased in both cases. Only ischemia, but not spreading depression, led to enhanced release of serine, threonine, and asparagine. We thus conclude that an elevation in the interstitial concentrations of non-neurotransmitter amino acids is specific to deep ischemic injury to nervous tissue. We propose the monitoring of serine, asparagine, and threonine, together with excitatory amino acids, as an index of the degree of ischemic brain injury.

Effects of 5-Day Styrene Inhalation on Serum LH and Testosterone Levels and on Hypothalamic and Striatal Amino Acid Neurotransmitter Concentrations in Male Rats

Effects of 5-Day Styrene Inhalation on Serum LH and Testosterone Levels and on Hypothalamic and Striatal Amino Acid Neurotransmitter Concentrations in Male Rats

Selective amino acids changes in the medial and lateral preoptic area in the formalin test in rats

A combination of microdialysis in freely moving rats and capillary zone electrophoresis coupled to laser induced fluorescence detection was used to measure extracellular concentrations of amino acid neurotransmitters in different hypothalamic areas during noxious stimulation. Arginine, glutamate and aspartate were monitored every 30 s before and after a s.c. injection of formalin (5%, 50 μl) or saline (0.9%) in the right hind paw. In the medial and lateral preoptic area, calcium and nerve impulse dependent increases of arginine, glutamate and aspartate were observed during the first 2 min after formalin injection. However, amino acid changes were not detected in the lateral hypothalamus or in the ventromedial nucleus when compared with pre-injection levels or with the levels from animals injected with saline in the hind paw. Flinching behavior was also scored during the first 10 min following the formalin or saline injection. Flinching frequency was maximum at minute 2 after formalin injection, whereas saline injection did not elicited any flinching behavior. These results show that nociceptive stimulation induces rapid and differential amino acids changes in discrete areas of the hypothalamus that can be associated with pain-related behavior.

Effects of 5-Day Styrene Inhalation on Serum LH and Testosterone Levels and on Hypothalamic and Striatal Amino Acid Neurotransmitter Concentrations in Male Rats

The volatile chemical styrene may impair male fertility. Testicular testosterone (T) production is controlled by the hypothalamic/pituitary/gonadal axis. From the mediobasal hypothalamus (MBH), gonadotropin-releasing hormone (GnRH) is released, which stimulates luteinizing hormone (LH) secretion from the pituitary, which in turn enhances T production. GnRH release is controlled by glutamate (GLU) and gamma-aminobutyric acid (GABA). GLU and GABA neurons are regulated by T. Thus, reduced fertility of styrene-exposed male workers may result from altered GLU/GABA neurotransmission, causing insufficient GnRH, LH, and T secretion. Therefore, we compared LH and T levels of male rats that have inhaled styrene (0, 150, 500, 1500 ppm for 6 h on 5 consecutive days) to GLU and GABA concentrations in the MBH and striatum. Animals were killed directly following the last exposure (immediate group) or after 24 h (recovery group). No suppression of LH or T levels was observed after styrene inhalation. LH levels of the immediate groups with 500 or 1500 ppm exposure were slightly but signficantly elevated. Hypothalamic GLU and GABA concentrations remained unchanged. Increased striatal GABA concentrations were determined in recovery groups with 500 or 1500 ppm exposure. Striatal GLU concentrations remained unaffected. Thus, we demonstrate slightly increased LH and T levels in styrene-exposed male rats after inhalation of the two higher doses. This effect did not correlate with hypothalamic GLU and GABA concentrations. With the limitations inherent to any animal model, these data obtained from a 5-day exposure study with rats suggest, but do not unequivocally prove, that styrene may have also no reproductive toxicity effects in men chronically exposed to this chemical.

Effects of chronic lithium and sodium valproate on concentrations of brain amino acids

The present study was designed to determine if the mood stabilizers, lithium and valproate, have common effects on concentrations of amino acid neurotransmitters which may be related to their mechanisms of action. Two separate groups of rats were administered therapeutic doses of lithium, sodium valproate, or saline for 2 weeks. Whole brain extracts were then examined using either high-field 1H NMR spectroscopy or HPLC. Both drugs decreased whole brain concentrations of aspartate, glutamate, and taurine while brain concentrations of γ-aminobutyric acid (GABA) and alanine decreased following chronic sodium valproate administration but not following chronic lithium administration. These findings indicate that lithium and sodium valproate share common effects on the concentrations of certain amino acid neurotransmitters in whole brain which may be related to their mechanisms of action in bipolar disorder.

Pharmacological effects of extracts from Valeriana adscendens Trel. II. Effects on GABA uptake and amino acids.

Methanolic and aqueous extracts from Valeriana adscendens (Valerianaceae), that previously demonstrated neuroleptic-like properties, were studied for their effects on GABA uptake and amino acid neurotransmitter levels. The methanolic estract showed a significant effect in inhibiting GABA uptake and in decreasing the intracellular content of amino acid neurotransmitters in crude synaptosomes of rat. The data obtained could explain the neuroleptic activity of methanolic extract of the plant and confirm its traditional use.

Analysis of extracellular γ-aminobutyric acid, glutamate and aspartate in cat visual cortex by in vivo microdialysis and capillary electrophoresis-laser induced fluorescence detection

To investigate the influence of a partial sensory deprivation on the extracellular concentration of amino acid neurotransmitters in cat visual cortex, a capillary electrophoresis method was developed for the quantification of γ-aminobutyric acid (GABA), glutamate (Glu) and aspartate (Asp) in in vivo microdialysis samples of cat brain. Microdialysis samples from different regions of area 17 were obtained every 15-min using CMA 12 2-mm probes perfused with synthetic cerebrospinal fluid and derivatized using fluorescein isothiocyanate (FITC). Laser-induced fluorescence (LIF) detection was employed. Good selectivity was obtained with a borate buffer (20 mM, pH 10.25). The whole procedure, including the washing step takes only 15 min. The conditions for derivatization and separation were optimized. The parameters for validation such as linearity, precision and detection limit are also reported. The results are consistent with those of HPLC but, as the sample volumes needed are only 1–5 nl, a much better time resolution can be obtained.

776 The Effect of Deep Brain Stimulation on the Concentration of Amino Acid Neurotransmitters in a Rat Model of Parkinson's Disease

776 The Effect of Deep Brain Stimulation on the Concentration of Amino Acid Neurotransmitters in a Rat Model of Parkinson's Disease

Glycine and other neurotransmitter amino acids in cerebrospinal fluid in perinatal asphyxia and neonatal hypoxic‐ischaemic encephalopathy

The aim of this study was to evaluate the concentrations of neurotransmitter amino acids in the cerebrospinal fluid (CSF) of asphyctic newborns, and to establish whether these concentrations are related to the degree of hypoxic‐ischaemic encephalopathy (HIE). Levels of glutamate, aspartate, glycine and taurine in CSF were measured in 48 full‐term newborns during the first 2 d of life. Thirty‐nine of these newborns had birth asphyxia and were divided into three groups: 11 without HIE, 19 with mild HIE and 9 with moderate HIE. None suffered from severe HIE. They were compared with a control group of 9 non‐hypoxic newborns. Determinations of the amino acids in CSF were made by chromatography and expressed as μmol/1 (mean ± SD). CSF glycine value was related to erythrocyte count, and CSF taurine value was related to its plasmatic level. Levels of CSF glycine were related to the severity of HIE (p= 0.020): control (12.08 ± 4.04 μmol/1), without HIE (12.49 ± 4.04 μmol/1), mild HIE (12.14 ± 3.83 μmol/1), and moderate HIE (19.1 ± 10.08 μmol/1; p<0.05 versus mild HIE), even after removing the influence of red cells in CSF.

Determination of submicromolar concentrations of neurotransmitter amino acids by fluorescence detection using a modification of the 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate method for amino acid analysis

A sensitive method for quantitatively determining submicromolar levels of neurotransmitter amino acids (e.g. Asp, Glu and γ-aminobutyric acid) in microdialysates from brain and cerebrospinal fluids is reported. 6-Aminoquinolyl- N-hydroxysuccinimidyl carbamate (AQC) was employed as the derivatization reagent, followed by HPLC separation and fluorescence detection of the derivatives. The derivatization was conducted simply by mixing the AQC directly with the microdialysis samples. The reaction was complete within seconds after mixing at room temperature. Separation development optimizing the gradient profile, eluent pH and column temperature resulted in an excellent separation of the required amino acids in less than 30 min. Other resolved amino acids in the same profile include Gly, taurine, and Pro. Recoveries for the amino acids of interest spiked into high salt containing perfusion buffers were greater than 97%. The sensitivity of the method was increased by employing a 16-μl flow cell in the detector and analyzing 20-μl aliquots of the derivatization mixtures. With the optimized conditions, the detection limits were 3–7 n M (fmol/μl). Typical reproducibility (%R.S.D.) for quantitation of these amino acids at submicromolar levels was approximately 2%. Excellent linearity ( r 2>0.999) was achieved over the range 0.2–20 μ M. The low detection limits permitted the analysis of a number of different microdialysate samples including those from cerebrospinal fluid, as well as substantia nigra and hypothalamus from brain samples, even at basal levels where γ-aminobutyric acid concentration may be <50 n M. The excellent sensitivity made it easy to distinguish basal from stimulated levels of neurotransmitter amino acids, even from sample sizes as small as 10 μl.

Plasma levels of neuroexcitatory amino acids in patients with migraine or tension headache

Plasma amino acids were analysed in patients with migraine with (9) and without (80) aura, in patients with tension headache (14) and in controls (62). The neuroexcitatory amino acids glutamic acid, glutamine, glycine, cysteic acid and homocysteic acid were elevated in migraine patients while total thiols (cysteine/cystine) were reduced. Patients with tension headache had values which were similar to those of controls. Tryptophan was elevated in migraine patients without aura only. Studies on two patients showed that the raised resting excitatory amino acid levels became still further elevated during a migraine attack. These results show that high concentrations of neurotransmitter amino acids occur normally in migraine patients and suggest that this profile may be a contributory factor in migraine attacks. Tension headache, however, has different biochemical parameters.

Construction of 1 mm microdialysis probe for amino acids dialysis in rats

We describe here a microdialysis probe with 1 mm opening for precise and confined dialysis area in the awake, freely moving rat. This probe is designed to allow the local diffusion of the perfusion medium to an area approximately 175 μm high, 266 μm wide (mediolateral direction), and 305 μm in rostrocaudal direction. In addition, the probe allows the local application of drugs to the same precise area of interest. The probe was constructed from a piece of 25 gauge tubing with 1 mm hallowed opening located 0.5 mm from the distal (inserting) end. The dialysis fiber which was inserted into the stainless steel 25 gauge tubing and cemented into place has 200 μm diameter and 5000 molecular weight cut off. We tested the probe diffusion extent by direct infusion of fluorogold through the dialysis cannula. Changes in the extracellular concentrations of amino acids were measured in response to infusion of veratridine a sodium channel activator. All amino acids tested showed a significant 80% times decrease in their recovery concentration when compared to their respective concentrations recovered through 2 mm probe constructed earlier in our laboratory ( Renno et al., 1992). Tests in awake rats with probes in the ventrocaudal PAG showed stable amounts of 12 different amino acids during repeated (6–8 times) 12 min samples at 3–5 μl/min collecting rate. Depolarization with 75 μM veratridine resulted in significant elevation in extracellular γ-aminobutyric acid (GABA), aspartate, glutamate, taurine, glycine and citrulline. This design enables us to apply drugs of interest and measure the concentrations of amino acid neurotransmitters to a more precise, delineated and premeasured areas in the CNS.

The GABAergic control of gonadotropin-releasing hormone secretion in male rats during sexual maturation involves effects on hypothalamic excitatory and inhibitory amino acid systems.

In order to evaluate the possible participation of the hypothalamic excitatory and inhibitory amino acid neurotransmitter systems in the GnRH release response to GABAergic drugs, hypothalami (preoptic and mediobasal area) of immature (26 days of age) and adult male rats were perifused with GABA-A and -B agonists and antagonists. GnRH and amino acid neurotransmitter concentrations (glutamate, taurine, GABA) were measured in perfusate samples collected every 15 min during 150 min. In immature rats, muscimol and baclofen (GABA-A and GABA-B agonists, respectively) increased GnRH, glutamate and GABA release and decreased taurine output, while in adults these agonists showed opposite effects on GnRH and glutamate release, and increased GABA and taurine output. On the other hand, in immature rats bicuculline and phaclofen (GABA-A and GABA-B antagonists, respectively) decreased GnRH, glutamate and GABA release, increasing taurine outflow. In adult animals, these antagonists enhanced GnRH and glutamate release, decreasing taurine and GABA outflow. These results indicate that GABA stimulates GnRH release in immature male rats and confirm the inhibitory role of this amino acid neurotransmitter in adult animals. This effect might be associated, at least partially, with the modifications observed in the excitatory and inhibitory amino acid release. On the other hand, in immature rats, stimulation of GABA-A and GABA-B receptors increased GABA release. Although ultrastructural studies have not produced any evidence of GABA-GABA neurointeractions, our results suggest the existence of a positive feedback mechanism of GABA autoregulation active during the prepubertal stage. Participation of this mechanism in the onset of puberty cannot be discarded.

Changes in Cerebellar Amino Acid Neurotransmitter Concentrations and Receptors Following Administration of the NeurotoxinL-2-Chloropropionic Acid

We studied the effect ofL-2-chloropropionic acid (L-CPA)-induced (250 mg/kg/po/day for 3 days) neurotoxicity, which results in an almost total destruction of cerebellar granule cells over 5 days, on forebrain and cerebellar neurochemistry. There was a reduction in cerebellar aspartate and glutamate concentrations of L-CPA-treated rats and a reduction inN-methyl-D-aspartate (NMDA) and kainate receptor densities in the cerebellar cortex following loss of the granule cells. Concentrations of glutamine and GABA were increased transiently during the development of the granule cell lesion but fell back to control levels by Day 5 of the study. Glycine concentrations began to rise as the granule cells began to disappear and concentrations remained elevated until the end of the study. In contrast, concentrations of taurine began to fall around the same time point as the granule cells were gradually depleted. We did not observe any consistent changes in forebrain amino acid concentrations following the L-CPA administration or any changes in NMDA, kainate, GABAA, or A1-adenosine receptor densities. We therefore conclude that the L-CPA-induced loss in cerebellar granule cells is accompanied by a reduction in cerebellar aspartate and glutamate concentrations and in the density of NMDA and kainate receptors in the cerebellar cortex. Changes in cerebellar GABA, glutamine, glycine, and taurine concentrations probably reflect secondary compensatory changes in cerebellar activity resulting from a widespread loss of cerebellar granule cells and loss of excitatory inputs. We suggest that L-CPA-induced neurotoxicity may be valuable tool to study cerebellar neurochemistry and physiology.

Intrastriatal injections of the succinate dehydrogenase inhibitor, malonate, cause a rise in extracellular amino acids that is blocked by MK-801

The effects of intrastriatal injections of a reversible inhibitor of succinate dehydrogenase, malonate, on the extracellular concentrations of amino acid neurotransmitters were examined using a microdialysis probe that was positioned a fixed distance from an injection cannula. Malonate (2 μmol) caused a 23 ± 5-fold increase in extracellular glutamate (Glu), a 18 ± 6-fold increase in extracellular γ-aminobutyric acid (GABA) and a modest increase in extracellular aspartate (Asp, 2.9 ± 0.8-fold increase). Administration of the NMDA receptor antagonist MK-801 (5 mg/kg) prior to injection of malonate almost completely blocked these increases. This study provides direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular amino acid neurotransmitters and further evidence that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through an excitotoxic mechanism.

Determination of in vivo amino acid neurotransmitters by high-performance liquid chromatography with o-phthalaldehyde-sulphite derivatisation

The measurement of amino acid neurotransmitters by high-performance liquid chromatography (HPLC) has emerged as a reliable and sensitive method. This paper describes a method which employs electrochemical (EC) detection of amino acid derivatives formed by a reaction with o-phthalaldehyde (OPA) in the presence of sulphite ions. This is discussed in relation to the problems of previously reported methods based on OPA derivatisation. Precise separation of the following 7 amino acid standards is achieved using isocratic elution: serine, glycine, taurine, glutamate, arginine, alanine and GABA, in order of increasing retention time. Total elution time is 25 min. Derivatisation proceeds at room temperature and the derivatives are stable for up to 5 h. This technique has the sensitivity to determine the concentrations of amino acid neurotransmitters in cerebrospinal fluid (CSF) and an in vivo microdialysis method is discussed for the detection of basal and potassium-stimulated levels of γ-aminobutyric acid and glutamate from rat hippocampus.

Excitatory Amino Acid Neurotransmitters in Human Cerebrospinal Fluid After Cardiopulmonary Resuscitation

Neurologic deficits are a major source of serious morbidity in patients resuscitated from cardiac arrest. Recent evidences support the hypothesis that accumulation of excitatory amino acids and overstimulation of their receptors in the brain tissue contribute to ischemic neuronal damage. In the present study, we measured concentrations of amino acid neurotransmitters, including glutamate and aspartate, in human cerebrospinal fluid (CSF) following cardiopulmonary resuscitation (CPR) to clarify whether CSF levels of excitatory amino acids reflect the global ischemic insults during cardiac arrest. In 11 post-CPR patients without any evidence of intracranial causes of cardiac arrest (the CPR group), CSF was sampled 2 to 12 hours following CPR. After deproteinization and storage at-80°C, derivatization, elution, and analysis of amino acids in CSF samples were performed by high-performance liquid chromatography. Sixteen patients scheduled for spinal anesthesia and elective surgery were regarded as normal control (the control group).The results showed that glutamate and aspartate in the CSF of the CPR group significantly increased 3.6 and 5.7-fold com pared with fhose the control group. Taurine and alanine also significantly increased. Thus, in the present clinical study, we demonstrated elevations of neurotoxic glutamate and aspartate in CSF following CPR, which might relate post-CPR neurologic deficits.

Hepatic encephalopathy in thioacetamide-induced acute liver failure in rats: Characterization of an improved model and study of amino acid-ergic neurotransmission†

An imbalance of excitatory and inhibitory amino acid-ergic neurotransmission has been suggested to play a role in the pathogenesis of hepatic encephalopathy. For further evaluation of this hypothesis, several parameters of amino acid-ergic neurotransmission were studied in rats with acute liver failure induced by the administration of 300 mg per kg thioacetamide by gavage on two consecutive days. By appropriate supportive care, hypoglycemia, renal failure and hypothermia were avoided. Rats were monitored clinically and neurologically. Hepatic encephalopathy evolved in four distinct, easily recognizable stages. Light and electron microscopic examination of brains of rats with hepatic encephalopathy revealed only a slight swelling of nuclei of neurons and astrocytes without signs of neuronal degeneration or brain edema. In rats with hepatic encephalopathy, the concentrations of GABA, glutamate and taurine were decreased in the cerebral cortex, the hippocampus and the striatum, whereas those of aspartate and glycine were unchanged or increased. GABAA and benzodiazepine receptors were studied as parameters for the postsynaptic GABAA-benzodiazepine receptor complex, glutamic acid decarboxylase as parameter for presynaptic GABA-ergic neurons and stimulation of benzodiazepine binding by GABA as a parameter for a GABA-mediated postsynaptic event. None of these parameters was different in hepatic encephalopathy as compared to controls. Similarly, Ca++/Cl(-)-dependent and -independent glutamate receptors as parameters for glutamatergic neurons were unchanged in rats with hepatic encephalopathy. Thus, in rats with thioacetamide-induced liver failure and hepatic encephalopathy, changes of the concentrations of neurotransmitter amino acids occur in the brain. Other neurochemical parameters, however, failed to identify alterations of GABA-ergic or glutamatergic neurotransmission in hepatic encephalopathy.

Amino acid and neuropeptide neurotransmitters in Huntington's disease cerebellum

Several neuropathologic studies have suggested that there may be pathologic involvement of the cerebellum in Huntington's disease (HD). To investigate this further, we measured concentrations of neurotransmitter amino acids and the neuropeptides, somatostatin, neuropeptide Y and substance P, in HD cerebellar cortex and dentate nucleus. Twenty-seven pathologically confirmed cases of HD were compared with 20 controls. There were no significant changes in concentrations of glutamate, alanine, ethanolamine, taurine or GABA in cerebellar cortex and dentate nucleus. Aspartate concentrations were significantly increased by 21% in HD cerebellar cortex. In the dentate nucleus, there were small significant increases of neuropeptide Y-like immunoreactivity and substance P-like immunoreactivity. The meaning of the neurotransmitter changes found is unclear: however, the lack of change in GABA and glutamate concentrations argues against a substantial loss of intrinsic cerebellar neurons.

Amino acid neurotransmitters in postmortem human brain analyzed by high performance liquid chromatography with electrochemical detection

In the present study we have developed a method of measuring putative neurotransmitter amino acids using high performance liquid chromatography (HPLC) with electrochemical detection. The assay had a sensitivity in the low pmol range and sample turnover time was 30 min. The postmortem stability of amino acids was examined in an animal model simulating human autopsy conditions. Aspartate concentrations increased 15% between 4 and 24 h postmortem while γ-aminobutyric acid (GABA) concentrations rose 35% by 4 h but were stable thereafter. Glutamate and taurine were stable at all time points. The assay has been used to examine concentrations of neurotransmitter amino acids in 15 patients without neurological or psychiatric disease. Results agree well with previous work and knowledge of amino acid neurotransmitter pathways. The current technique provides a reliable method for the study of amino acid transmitter abnormalities in neurological illness.