Glutamate And Γ-aminobutyric Acid Research Articles

Optimization of ultra-performance liquid chromatography (UPLC) with fluorescence detector (FLD) method for the quantitative determination of selected neurotransmitters in rat brain.

Glutamate (Glu) and γ-aminobutyric acid (GABA) are the main neurotransmitters in the central nervous system for excitatory and inhibitory processes, respectively. Monitoring these neurotransmitters is an essential tool in establishing pathological functions, among others in terms of occupational exposure to toxic substances. We present modification of the HPLC (high-performance liquid chromatography) to the UPLC (ultra-performance liquid chromatography) method for the simultaneous determination of glutamate and γ-aminobutyric acid in a single injection. The isocratic separation of these neurotransmitter derivatives was performed on Waters Acquity BEH (ethylene bridged hybrid) C18 column with particle size of 1.7 μm at 35°C using a mobile phase consisting of 0.1 M acetate buffer (pH 6.0) and methanol (60:40, v/v) at a flow rate of 0.3 ml/min. The analytes were detected with the fluorescence detector (FLD) using derivatization with o-phthaldialdehyde (OPA), resulting in excitation at 340 nm and emission at 455 nm. Several validation parameters including linearity (0.999), accuracy (101.1%), intra-day precision (1.52-1.84%), inter-day precision (2.47-3.12%), limit of detection (5-30 ng/ml) and quantification (100 ng/ml) were examined. The developed method was also used for the determination of these neurotransmitters in homogenates of selected rat brain structures. The presented UPLC-FLD is characterized by shorter separation time (3.5 min), which is an adaptation of the similar HPLC methods and is an alternative for more expensive references techniques such as liquid chromatography coupled with tandem mass-spectrometry (LC-MS/MS) methods. Med Pr 2017;68(5):583-591.

Developmental vitamin D deficiency alters multiple neurotransmitter systems in the neonatal rat brain

BackgroundEpidemiological evidence suggests that developmental vitamin D (DVD) deficiency is a risk factor for neuropsychiatric disorders, such as schizophrenia. DVD deficiency in rats is associated with altered brain structure and adult behaviours indicating alterations in dopamine and glutamate signalling. Developmental alterations in dopamine neurotransmission have also been observed in DVD-deficient rats but a comprehensive assessment of brain neurochemistry has not been undertaken. Thus, the current study determined the regional concentrations of dopamine, noradrenaline, serotonin, glutamine, glutamate and γ-aminobutyric acid (GABA), and associated metabolites, in DVD-deficient neonates. MethodsSprague-Dawley rats were fed a vitamin D deficient diet or control diet six weeks prior to mating until birth and housed under UVB-free lighting conditions. Neurotransmitter concentration was assessed by high-performance liquid chromatography on post-mortem neonatal brain tissue. ResultsUbiquitous reductions in the levels of glutamine (12–24%) were observed in DVD-deficient neonates compared with control neonates. Similarly, in multiple brain regions DVD-deficient neonates had increased levels of noradrenaline and serine compared with control neonates. In contrast, increased levels of dopamine and decreased levels of serotonin in DVD-deficient neonates were limited to striatal subregions compared with controls. ConclusionsOur results confirm that DVD deficiency leads to changes in multiple neurotransmitter systems in the neonate brain. Importantly, this regionally-based assessment in DVD-deficient neonates identified both widespread neurotransmitter changes (glutamine/noradrenaline) and regionally selective neurotransmitter changes (dopamine/serotonin). Thus, vitamin D may have both general and local actions depending on the neurotransmitter system being investigated. Taken together, these data suggest that DVD deficiency alters neurotransmitter systems relevant to schizophrenia in the developing rat brain.

A sensitive and practical RP-HPLC-FLD for determination of the low neuroactive amino acid levels in body fluids and its application in depression

Ion-exchange high performance liquid chromatography (HPLC) generally fails as a method to determine low levels of free amino acids (AAs) in body fluids. Here we present a modified reversed-phase HPLC (RP-HPLC) protocol for the determination of AAs in body fluids and its application in mood disorder patients. We improved a previous research protocol by modifying i) sample preparation, including deproteination, ii) derivitization, including derivating agent and condition, and iii) sample separation, which is mainly determined by the pH value, the components and the additives of the mobile phases. The combination of these modifications, together with fluorescence detection (FLD), allows sensitive and practical determination of free AA levels in body fluids of depressive patients. This protocol was validated by determining the postmortem cerebrospinal fluid (CSF) glutamic acid (Glu) and γ-aminobutyric acid (GABA) levels of 8 major depressive disorder (MDD) patients, 9 bipolar disorder (BD) patients, and 19 well-matched controls, while also testing the plasma and CSF AA levels of living MDD patients. CSF Glu and GABA levels were both significantly decreased in MDD but not in BD patients. The data indicate that this RP-HPLC-FLD protocol is applicable for detection of low levels of neuroactive AAs in body fluids, as well as for routine clinical applications.

Extraction methods and sedative–hypnotic effects of polysaccharide and total flavonoids of Cordyceps militaris

ABSTRACTSedatives and hypnotics made from Chinese herbal medicine show great market prospects for minor side effects and zero potential addiction. In this study, the extraction conditions of Cordyceps militaris polysaccharide (CMP) and flavonoids (CMF) were, respectively, optimized by orthogonal experiments as follows: 30:1 ratio of water to plant material, three rounds of extraction at 90 °C and 3 h for each extraction; 40:1 ratio of 70% ethanol to plant material, reflux extraction at 90 °C for 4 h. Then, 200, 100 and 50 mg/kg of CMP and CMF were given intragastrically to mice for 30 days. The locomotor activity times of the mice were recorded on the 14th and 30th days. The effects of CMP and CMF on the sleep induced by pentobarbital sodium were observed on the 30th day. 5-Hydroxytryptamine (5-HT), acetylcholine (ACh), glutamate (Glu) and γ-aminobutyric acid (GABA) levels in the mouse brains were determined by enzyme-linked immunosorbent assay on the 30th day. The results showed that different doses of CMP and CMF could reduce the number of locomotor activities in mice and lower the Glu level (p < 0.05 in the 50 mg/kg CMP group and p < 0.01 in the other groups), elevate the 5-HT level and reduce the ACh level (p < 0.01 only in the 200 mg/kg CMP and CMF groups) in the mouse brains. Therefore, the elevated 5-HT levels and decreased ACh and Glu levels in the brains may be the main mechanisms through which CMP and CMF exert their sedative and hypnotic effects.

Migraine and magnetic resonance spectroscopy: a systematic review.

To present an updated and streamlined overview of the metabolic and biochemical aspect of the migraine pathophysiology based on findings from phosphorous (P) and hydrogen (H) magnetic resonance spectroscopy (MRS) studies. Despite of the variation in the methodology and quality of the MRS migraine studies over time, some results were consistent and reproducible. P-MRS studies suggested reduced availability of neuronal energy and implied a mitochondrial dysfunction in the migraine brain. H-MRS studies reported interictal abnormalities in the excitatory and inhibitory neurotransmitters, glutamate and γ-aminobutyric acid (GABA), suggesting persistent altered excitability in migraine patients. N-Acetylaspartate levels were decreased in migraine, probably due to a mitochondrial dysfunction and abnormal energy metabolism. The reported abnormalities may increase the susceptibility of migraine patients to excitatory stimulation, such as migraine attack triggers. Several biochemical aspects of the migraine pathophysiology remain to be elucidated using MRS, such as the migraine attack, correlation to disease severity, and medication efficacy. Nevertheless, to identify a biomarker in migraine, MRS may be a valuable noninvasive technique.

Impact of dexmedetomidine on amino acid contents and the cerebral ultrastructure of rats with cerebral ischemia-reperfusion injury

To investigate the effects of dexmedetomidine (DEX) on amino acid contents and the cerebral ultrastructure of rats with cerebral ischemia-reperfusion injury (I/R). Thirty-six, male, Wistar rats were randomly divided into three groups: the sham operation group (group C), the ischemia-reperfusion group (group I/R), and the DEX group (group D). The middle cerebral artery occlusion model was prepared by the modified Longa method. The time of ischemia was 180 min, and 120 min after reperfusion, the amount of glutamate (Glu), and γ-aminobutyric acid (GABA) in the brain were measured, and the ultrastructure-level changes in the cerebral cortex were examined using electron microscopy. Compared to group C, Glu contents in group D, and I/R significantly increased. Compared to group I/R, Glu contents in group D significantly decreased. Compared to group C, GABA contents in group D, and I/R significantly increased, and those in group D significantly increased, as compared to group I/R. The cerebral ultrastructure was normal in group C. Vacuolar degeneration in the plastiosome and nervous processes, was more critical than in group D. Vascular endothelial cells (VEC) were damaged. On the contrary, these changes in group D significantly improved. Dexmedetomidine is capable of decreasing glutamergic content, and increasing GABAergic content, in order to decrease the injury of the cerebral ultrastructure, following cerebral ischemia-reperfusion injury.

Sedentary conditions and enhanced responses to GABA in the RVLM: role of the contralateral RVLM.

A sedentary lifestyle is a major risk factor for cardiovascular disease, and both conditions are associated with overactivity of the sympathetic nervous system. Ongoing discharge of sympathetic nerves is regulated by the rostral ventrolateral medulla (RVLM), which in turn is modulated by the primary excitatory and inhibitory neurotransmitters glutamate and γ-amino-butyric acid (GABA), respectively. We reported previously that sedentary conditions enhance GABAergic modulation of sympathoexcitation in the RVLM, despite overall increased sympathoexcitation. Thus the purpose of this study was to test the hypothesis that sedentary conditions increase responsiveness to GABA in RVLM. Male Sprague-Dawley rats performed either chronic wheeling running or remained sedentary for 12-15 wk. Animals were instrumented to perform RVLM microinjections under Inactin anesthesia while mean arterial pressure (MAP) and splanchnic sympathetic nerve activity (SSNA) were recorded. Unilateral microinjections of GABA (30 nl, 0.3-600 mM) into the RVLM produced dose-dependent decreases in MAP and SSNA; however, no group differences were observed. Inhibition of the contralateral RVLM (muscimol, 2 mM, 90 nl) caused decreases in MAP and SSNA that were not different between groups but enhanced decreases in SSNA to GABA in sedentary rats only. In sinoaortic denervated rats, GABA microinjections before or after inhibition of the contralateral RVLM caused decreases in MAP and SSNA that were not different between groups. Our results suggest that the contralateral RVLM plays an important role in buffering responses to inhibition of the ipsilateral RVLM under sedentary but not physically active conditions. Based on these studies and others, sedentary conditions appear to enhance both sympathoinhibitory and sympathoexcitatory mechanisms in the RVLM. Enhanced sympathoinhibition may act to reduce already elevated sympathetic nervous system activity following sedentary conditions.

Parachlorophenylalanine-induced 5-HT depletion alters behavioral and brain neurotransmitters levels in 6-Hz psychomotor seizure model in mice.

The present study was designed to investigate the role of serotonin and other neurotransmitters namely dopamine (DA), histamine, nor-epinephrine (NE), glutamate, and γ-aminobutyric acid (GABA) in the 6-Hz-induced psychomotor seizures in Swiss albino mice. Parachlorophenylalanine (PCPA, 300 mg/kg/day, i.p for 3 days)-treated mice were given 6-Hz stimulation. Sodium valproate (SVP) (200 mg/kg/day, p.o for 3 days) was used as a reference antiepileptic drug. The behavioral changes induced by 6 Hz including increased rearing and grooming, Straub's tail, behavioral arrest, stun position were amplified by PCPA. The 6-Hz-induced seizures were accompanied by reduced brain 5-HT, DA, NE, histamine, GABA, and enhanced glutamate levels. PCPA facilitated further reduction of endogenous 5-HT and DA levels but not NE, histamine, GABA, and glutamate levels. Pre- and post-treatment with SVP protected the mice from 6-Hz seizures and attenuated PCPA-induced changes in the levels of 5-HT and DA in the mice brain suggesting the protective effect of SVP in the pharmacoresistant model of epilepsy involving mainly serotonergic mechanism. However, the study also suggests modulation of other neurotransmitters both in 6-Hz psychomotor seizures and in the action of SVP against such seizures.

A Novel Bio-Psychosocial-Behavioral Treatment Model in Schizophrenia.

Despite the substantial burden of illness in schizophrenia, there has been a discrepancy between the beneficial effects of an increased use of antipsychotic medications and achieving limited recovery or remission. Because the focus of the most common antipsychotic medications is on dopamine, which is associated with positive symptoms, there is an unmet need for patients with negative symptoms. Since cognitive and negative symptoms rather than positive symptoms are more closely associated with psychosocial impairments in patients with schizophrenia, the non-dopaminergic systems including glutamate and γ-aminobutyric acid (GABA) of the prefrontal cortex should be of concern as well. The balance of excitation and inhibition has been associated with epigenetic modifications and thus can be analyzed in terms of a neurodevelopmental and neural circuitry perspective. Hence, a novel bio-psychosocial-behavioral model for the treatment of schizophrenia is needed to account for the non-dopaminergic systems involved in schizophrenia, rather than dopaminergic mechanisms. This model can be understood from the viewpoint of neurodevelopment and neural circuitry and should include the staging care, personalized care, preventive care, reducing the cognitive deficits, and reducing stigma. Thomas R. Insel proposed this as a goal for schizophrenia treatment to be achieved by 2030.

In vivo evaluation of the hippocampal glutamate, GABA and the BDNF levels associated with spatial memory performance in a rodent model of neuropathic pain

BackgroundPatients with chronic pain usually suffer from learning and memory impairment which may significantly decrease their quality of life. Despite laboratory and clinical studies, the mechanism underlying this memory impairment remains elusive. We evaluated the effect of chronic pain on the glutamate and GABA levels and BDNF expression in the CA1 region of hippocampus as a possible explanation for memory impairment related to neuropathic pain. MethodsIn this respect, 30 male rats were randomly allocated to 3 groups as control, sham and neuropathic. Neuropathic pain was induced by a chronic constriction injury of the sciatic nerve (CCI) and mechanical allodynia and the spatial memory was assessed using the Von Frey filaments and Morris water maze respectively. To determine the potential mechanisms, the in vivo extracellular levels of glutamate and γ-aminobutyric acid (GABA) were measured by microdialysis and the brain-derived neurotrophic factor (BDNF) expression was determined by using western blots technique in the hippocampus on days 14 and 21 post-CCI. ResultsWe showed that CCI impaired spatial learning and memory in Morris water maze (MWM) task. BDNF expression level and glutamate concentration significantly decreased in rats with chronic constriction injury of the sciatic nerve (P<0.001, F=7.3, F=23.23). In addition, GABA increased in hippocampal CA1 region (P<0.001, F=39.2) when the pain threshold was minimum. Nevertheless, these changes reversed while pain was relieved spontaneously. ConclusionChronic pain induced by constriction of the sciatic nerve impairs the spatial learning and memory function in rats. This effect exerts through the increase in GABA concentration and decrease in the glutamate and BDNF levels in the CA1 region of the hippocampus.

Alterations of Glutamate and γ-Aminobutyric Acid Expressions in Normal and Myopic Eye Development in Guinea Pigs.

The retina has an important role in the signal transmission related to eye development and myopia. Glutamate (Glu) and γ-aminobutyric acid (GABA) are the major excitatory and inhibitory neurotransmitters in the retina, which can affect the development of both the eye and myopia. Nevertheless, change in the balance between the excitatory and inhibitory neurotransmitters still is unclear during development of eyes and myopia. The purpose of this study was to explore the alterations of the ratio of Glu to GABA (RGG), which mediates the balance between the excitatory and inhibitory neurotransmitters in retina in the development of the eyes and lens-induced myopia (LIM) in a guinea pig model. An LIM guinea pig model was established using a -10 diopter (D) negative lens. The levels of Glu, GABA, and the dynamic change of RGG were measured in the retina in normal and myopia guinea pigs at four time points (i.e., 0, 2, 4, and 6 weeks after onset of LIM). Considering that Glu and GABA are related closely to the occurrence of myopia, we further studied the changes of RGG in the retina in LIM guinea pigs. Our results showed that the RGG was upregulated and was well correlated with diopter and axial length than either Glu or GABA during the development of normal eyes. Besides, we observed that the content of the RGG in the retina in myopia eyes was higher than that of Glu and GABA in normal subjects and was an obviously positive correlation. Taken together, our findings suggest that the RGG has a pivotal role in eye development and myopia. The abnormal retina signal induced by the unbalanced ratio between Glu and GABA is related closely to the occurrence of myopia.

Effect of Selective 5-HT6R Agonist on Expression of 5-HT Receptor and Neurotransmitter in Vascular Dementia Rats.

Background5-HT6 receptor (5-HT6R) has pluripotent roles regulating secretion of neurotransmitters. However, whether 5-HT6R is involved in the development of vascular dementia (VD) remains unclear. To evaluate the role and mechanism of 5-HT6R in VD, this study established a rat VD model to evaluate the effect of selective 5-HT6R agonist on the expression of 5-HT6R mRNA and neurotransmitter.Material/MethodsEighty healthy male SD rats (7 weeks old) were randomly assigned to sham, model, 5-HT6R agonist, and placebo groups (N=20 each). A rat VD model was generated by permeant bilateral ligation of the common carotid artery. 5-HT6R agonist, placebo, or saline were given intraperitoneally for 4 weeks. The Morris water maze was utilized to test learning and memory function. Brains were extracted to separate the cortex and hippocampal tissues, in which glutamate and γ-aminobutyric acid (GABA) levels were analyzed. mRNA and protein levels of 5-HT6R were determined by RT-PCR and immunohistochemistry (IHC), respectively.ResultsModel rats had longer escape latency and fewer crossing platform times. Contents of DA, Glu, GABA, and Ach were lowered in cortical and hippocampal tissues, and 5-HT6R expression was suppressed (p<0.05). The application of 5-HT6R agonist shortened escape latency and increased the number of passing through the platform. It also improved hippocampal CA1 neuronal damage and elevated DA, Glu, GABA, and Ach contents and expression of 5-HT6R. Expression of 5-HT6R was not different from the placebo group.ConclusionsSelective 5-HT6R agonist can alleviate learning deficit of VD rats, possibly via improving neurotransmitter levels in brain regions.

GABA and glutamate levels correlate with MTR and clinical disability: Insights from multiple sclerosis

Converging areas of research have implicated glutamate and γ-aminobutyric acid (GABA) as key players in neuronal signalling and other central functions. Further research is needed, however, to identify microstructural and behavioral links to regional variability in levels of these neurometabolites, particularly in the presence of demyelinating disease. Thus, we sought to investigate the extent to which regional glutamate and GABA levels are related to a neuroimaging marker of microstructural damage and to motor and cognitive performance. Twenty-one healthy volunteers and 47 people with multiple sclerosis (all right-handed) participated in this study. Motor and cognitive abilities were assessed with standard tests used in the study of multiple sclerosis. Proton magnetic resonance spectroscopy data were acquired from sensorimotor and parietal regions of the brains’ left cerebral hemisphere using a MEGA-PRESS sequence. Our analysis protocol for the spectroscopy data was designed to account for confounding factors that could contaminate the measurement of neurometabolite levels due to disease, such as the macromolecule signal, partial volume effects, and relaxation effects. Glutamate levels in both regions of interest were lower in people with multiple sclerosis. In the sensorimotor (though not the parietal) region, GABA concentration was higher in the multiple sclerosis group compared to controls. Lower magnetization transfer ratio within grey and white matter regions from which spectroscopy data were acquired was linked to neurometabolite levels. When adjusting for age, normalized brain volume, MTR, total N-acetylaspartate level, and glutamate level, significant relationships were found between lower sensorimotor GABA level and worse performance on several tests, including one of upper limb motor function. This work highlights important methodological considerations relevant to analysis of spectroscopy data, particularly in the afflicted human brain. These findings support that regional neurotransmitter levels are linked to local microstructural integrity and specific behavioral abilities that can be affected in diseases such as multiple sclerosis.

Recent advances in the medicinal chemistry of group II and group III mGlu receptors.

Metabotropic glutamate receptors (mGlu receptors) belong to the G-protein-coupled receptors superfamily. They are divided into three groups, in which group II and group III belong to presynaptic receptors that negatively modulate glutamate and γ-aminobutyric acid (GABA) release when activated. In this review, we introduce not only the functions of mGlu receptors, but also the group II and group III allosteric modulators and agonists/antagonists reported over the past five years according to a classification of their structures, with a specific focus on their biological activity and selectivity. In particular, the structure of these compounds and the future directions of ideal candidates are highlighted.

Organization and functions of mGlu and GABAB receptor complexes.

The neurotransmitters glutamate and γ-aminobutyric acid (GABA) transmit synaptic signals by activating fast-acting ligand-gated ion channels and more slowly acting G-protein-coupled receptors (GPCRs). The GPCRs for these neurotransmitters, metabotropic glutamate (mGlu) and GABAB receptors, are atypical GPCRs with a large extracellular domain and a mandatory dimeric structure. Recent studies have revealed how these receptors are activated through multiple allosteric interactions between subunit domains. It emerges that the molecular complexity of these receptors is further increased through association with trafficking, effector and regulatory proteins. The structure and composition of these receptors present opportunities for therapeutic intervention in mental health and neurological disorders.

Imbalance between Glutamate and GABA in Fmr1 Knockout Astrocytes Influences Neuronal Development.

Fragile X syndrome (FXS) is a form of inherited mental retardation that results from the absence of the fragile X mental retardation protein (FMRP), the product of the Fmr1 gene. Numerous studies have shown that FMRP expression in astrocytes is important in the development of FXS. Although astrocytes affect neuronal dendrite development in Fmr1 knockout (KO) mice, the factors released by astrocytes are still unclear. We cultured wild type (WT) cortical neurons in astrocyte-conditioned medium (ACM) from WT or Fmr1 KO mice. Immunocytochemistry and Western blotting were performed to detect the dendritic growth of both WT and KO neurons. We determined glutamate and γ-aminobutyric acid (GABA) levels using high-performance liquid chromatography (HPLC). The total neuronal dendritic length was reduced when cultured in the Fmr1 KO ACM. This neurotoxicity was triggered by an imbalanced release of glutamate and GABA from Fmr1 KO astrocytes. We found increased glutaminase and GABA transaminase (GABA-T) expression and decreased monoamine oxidase B expression in Fmr1 KO astrocytes. The elevated levels of glutamate contributed to oxidative stress in the cultured neurons. Vigabatrin (VGB), a GABA-T inhibitor, reversed the changes caused by glutamate and GABA release in Fmr1 KO astrocytes and the abnormal behaviors in Fmr1 KO mice. Our results indicate that the imbalance in the astrocytic glutamate and GABA release may be involved in the neuropathology and the underlying symptoms of FXS, and provides a therapeutic target for treatment.

Elevated basal glutamate and unchanged glutamine and GABA in refractory epilepsy: Microdialysis study of 79 patients at the yale epilepsy surgery program.

Aberrant glutamate and γ-aminobutyric acid (GABA) neurotransmission contribute to seizure generation and the epileptic state. However, whether levels of these neurochemicals are abnormal in epileptic patients is unknown. Here, we report on interictal levels of glutamate, glutamine, and GABA in epilepsy patients at seizure onset and nonepileptic sites, cortical lesions, and from patients with poorly localized neocortical epilepsies. Subjects (n = 79) were medically refractory epilepsy patients undergoing intracranial electroencephalogram evaluation. Microdialysis probes (n = 125) coupled to depth electrodes were implanted within suspected seizure onset sites and microdialysis samples were obtained during interictal periods. Glutamate, glutamine, and GABA were measured using high-performance liquid chromatography. Probe locations were subsequently classified by consensus of expert epileptologists. Glutamate levels were elevated in epileptogenic (p = 0.03; n = 7), nonlocalized (p < 0.001), and lesional cortical sites (p < 0.001) when compared to nonepileptogenic cortex. Glutamate was also elevated in epileptogenic (p < 0.001) compared to nonepileptogenic hippocampus. There were no statistical differences in GABA or glutamine, although GABA levels showed high variability across patients and groups. Our findings indicate that chronically elevated extracellular glutamate is a common pathological feature among epilepsies with different etiology. Contrary to our predictions, GABA and glutamine levels were not decreased in any of the measured areas. Whereas variability in GABA levels may in part be attributed to the use of GABAergic antiepileptic drugs, the stability in glutamine across patient groups indicate that extracellular glutamine levels are under tighter metabolic regulation than previously thought. Ann Neurol 2016;80:35-45.

Validated methods for determination of neurotransmitters and metabolites in rodent brain tissue and extracellular fluid by reversed phase UHPLC-MS/MS.

Fast and sensitive methods for simultaneous determination of dopamine (DA), the two DA-metabolites homovanillic acid (HVA) and 3-methoxytyramine (3-MT), serotonin (5-HT) and the 5-HT-metabolite 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE), acetylcholine (ACh), glutamic acid (Glu) and γ-aminobutyric acid (GABA) in rodent brain tissue (1.0-4000nM) and extracellular fluid (ECF) (0.5-2000nM) based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) have been developed. Of the three different sample preparation methods for brain tissue samples tested, a simple and rapid protein precipitation procedure with formic acid was found to give the best results. The neurotransmitters (NTs) and NT metabolites were separated using UHPLC with an Acquity UPLC HSS T3 C18 column (2.1×100mm, 1.8μm particle size) with acidic mobile phase. Gradient elution with methanol was used and quantification was performed using multiple reaction monitoring (MRM). The total run time was 5.2min including equilibration time. The methods were validated by determining calibration model, intra- and inter-day precision and accuracy, limit of detection (LOD), lower limit of quantification (LLOQ), matrix effects (ME), carry-over and stability. Surrogate analytes were used to enable determination of the recovery and ME of the endogenous analytes in brain tissue. The methods were applied for determination of NTs at basal levels in rodent brain ECF and brain tissue homogenate. The developed methods are valuable tools in the studies of mechanisms of drugs of abuse, and neurologic and psychiatric disease.

Combined glutamate and glutamine levels in pain-processing brain regions are associated with individual pain sensitivity.

The relationship between glutamate and γ-aminobutyric acid (GABA) levels in the living human brain and pain sensitivity is unknown. Combined glutamine/glutamate (Glx), as well as GABA levels can be measured in vivo with single-voxel proton magnetic resonance spectroscopy. In this cross-sectional study, we aimed at determining whether Glx and/or GABA levels in pain-related brain regions are associated with individual differences in pain sensitivity. Experimental heat, cold, and mechanical pain thresholds were obtained from 39 healthy, drug-free individuals (25 men) according to the quantitative sensory testing protocol and summarized into 1 composite measure of pain sensitivity. The Glx levels were measured using point-resolved spectroscopy at 3 T, within a network of pain-associated brain regions comprising the insula, the anterior cingulate cortex, the mid-cingulate cortex, the dorsolateral prefrontal cortex, and the thalamus. GABA levels were measured using GABA-edited spectroscopy (Mescher-Garwood point-resolved spectroscopy) within the insula, the anterior cingulate cortex, and the mid-cingulate cortex. Glx and/or GABA levels correlated positively across all brain regions. Gender, weekly alcohol consumption, and depressive symptoms were significantly associated with Glx and/or GABA levels. A linear regression analysis including all these factors indicated that Glx levels pooled across pain-related brain regions were positively associated with pain sensitivity, whereas no appreciable relationship with GABA was found. In sum, we show that the levels of the excitatory neurotransmitter glutamate and its precursor glutamine across pain-related brain regions are positively correlated with individual pain sensitivity. Future studies will have to determine whether our findings also apply to clinical populations.

Effect of Jian-Pi-Zhi-Dong Decoction on striatal glutamate and γ-aminobutyric acid levels detected using microdialysis in a rat model of Tourette syndrome.

BackgroundJian-Pi-Zhi-Dong Decoction (JPZDD) is a dedicated treatment of Tourette syndrome (TS). The balance of neurotransmitters in the cortico-striato-pallido-thalamo-cortical network is crucial to the occurrence of TS and related to its severity. This study evaluated the effect of JPZDD on glutamate (Glu) and γ-aminobutyric acid (GABA) and their receptors in a TS rat model.Materials and methodsRats were divided into four groups (n=12 each). TS was induced in three of the groups by injecting them with 3,3′-iminodipropionitrile for 7 consecutive days. Two model groups were treated with tiapride (Tia) or JPZDD, while the control and the remaining model group were gavaged with saline. Behavior was assessed by stereotypic score and autonomic activity. Striatal Glu and GABA contents were detected using microdialysis. Expressions of N-methyl-D-aspartate receptor 1 and GABAA receptor (GABAAR) were observed using Western blot and real-time polymerase chain reaction.ResultsTia and JPZDD groups had decreased stereotypy compared with model rats; however, the JPZDD group showed a larger decrease in stereotypy than the Tia group at a 4-week time point. In a spontaneous activity test, the total distance of the JPZDD and Tia groups was significantly decreased compared with the model group. The Glu levels of the model group were higher than the control group and decreased with Tia or JPZDD treatment. The GABA level was higher in the model group than the control group. Expressions of GABAAR protein in the model group were higher than in the control group. Treatment with Tia or JPZDD reduced the expression of GABAAR protein. In the case of the mRNA expression, only Tia reduced the expression of N-methyl-D-aspartate receptor 1, compared with the model group.ConclusionJPZDD could alleviate impairments in behavior and dysfunctional signaling by downregulating GABAAR in the striatum. We suggest that this acts to maintain the balance of Glu and GABA.