Extracellular Brain Glutamate Research Articles

The Relationship between Plasma Glutamate Levels and Sleep Quality in HIV Patients

Background: Disturbance sleep quality is often found in sufferers of human immunodeficiency virus (HIV). Disturbed sleep quality can affect immunity, which ultimately can increase patient morbidity and mortality. Impaired sleep quality in HIV sufferers is related to neurotoxicity due to the HIV virus, which damages sleep architecture. HIV infection can cause an increase in brain extracellular glutamate. Elevated glutamate plays a role in neuronal and glial damage and death. This study aimed to assess the relationship between plasma glutamate levels and sleep quality in HIV sufferers.
 Methods: The research uses a cross-sectional design. The samples were HIV sufferers in a polyclinic voluntary counseling test (VCT) internal medicine of Dr. M. Djamil General Hospital Padang, who met the inclusion and exclusion criteria. Samples are selected by consecutive methods. Sleep quality was assessed using a questionnaire called the Pittsburgh sleep quality index (PSQI). Plasma glutamate levels were measured using the ELISA method. Statistical analysis using SPSS with a p-value <0.05 was considered statistically significant.
 Results: The research sample consisted of 82 people. The median plasma glutamate level was 16.39 µg/mL. Impaired sleep quality was found in 45 (54.9%) HIV sufferers. There was no significant relationship between plasma glutamate levels (p= 0.506), age (p=0.795), gender (p=0.547), education (p=0.358), occupation (p=0.255), disease duration (p=0.348), stage (p=0.309) and type of ARV therapy (p=0.791) with sleep quality in HIV sufferers. From this research, a significant relationship was found between sleep quality, body mass index (BMI) (p= 0.015), and marital status (p= 0.039).
 Conclusion: There is no relationship between plasma glutamate levels and sleep quality in HIV sufferers. There are other factors that influence sleep quality, namely BMI and marital status.

Blood Glutamate Scavenging With Pyruvate as a Novel Preventative and Therapeutic Approach for Depressive-Like Behavior Following Traumatic Brain Injury in a Rat Model.

Depression is a common and serious complication following traumatic brain injury (TBI). Both depression and TBI have independently been associated with pathologically elevated extracellular brain glutamate levels. In the setting of TBI, blood glutamate scavenging with pyruvate has been widely shown as an effective method to provide neuroprotection by reducing blood glutamate and subsequent brain glutamate levels. Here we evaluate pyruvate as a novel approach in the treatment and prevention of post-TBI depression-like behavior in a rat model. Rats were divided into five groups: (1) sham-operated control with pyruvate, (2) sham-operated control with placebo, (3) post-TBI with placebo, (4) post-TBI given preventative pyruvate, and (5) post-TBI treated with pyruvate. These groups had an equal number of females and males. Rats were assessed for depressive-like behavior, neurological status, and glutamate levels in the blood and brain. Post-TBI neurological deficits with concurrent elevations in glutamate levels were demonstrated, with peak glutamate levels 24 h after TBI. Following TBI, the administration of either prophylactic or therapeutic pyruvate led to reduced glutamate levels, improved neurologic recovery, and improved depressive-like behavior. Glutamate scavenging with pyruvate may be an effective prophylactic and therapeutic option for post-TBI depression by reducing associated elevations in brain glutamate levels.

Increased branched-chain amino acids at baseline and hours before a spontaneous seizure in the human epileptic brain.

The objective of this study was to monitor the extracellular brain chemistry dynamics at baseline and in relation to spontaneous seizures in human patients with refractory epilepsy. Thirty patients with drug-resistant focal epilepsy underwent intracranial electroencephalography and concurrent brain microdialysis for up to 8 continuous days. Extracellular brain glutamate, glutamine, and the branched-chain amino acids (BCAAs) valine, leucine, and isoleucine were quantified in the dialysis samples by liquid chromatography-tandem mass spectrometry. Extracellular BCAAs and glutamate were chronically elevated at baseline by approximately 1.5-3-fold in brain regions of seizure onset and propagation versus regions not involved by seizures. Moreover, isoleucine increased significantly above baseline as early as 3h before a spontaneous seizure. BCAAs play important roles in glutamatergic neurotransmission, mitochondrial function, neurodegeneration, and mammalian target of rapamycin signaling. Because all of these processes have been implicated in epilepsy, the results suggest a novel role of BCAAs in the pathogenesis of spontaneous seizures.

Circadian-Like Rhythmicity of Extracellular Brain Glutamate in Epilepsy.

Seizures often exhibit striking circadian-like (~24-h) rhythms. While chronotherapy has shown promise in treating epilepsy, it is not widely used, in part because the patterns of seizure rhythmicity vary considerably among patients and types of epilepsy. A better understanding of the mechanisms underlying rhythmicity in epilepsy could be expected to result in more effective approaches which can be tailored to each individual patient. The excitatory neurotransmitter glutamate is an essential modulator of circadian rhythms, and changes in the extracellular levels of glutamate likely affect the threshold to seizures. We used a reverse translational rodent model of mesial temporal lobe epilepsy (MTLE) combined with long-term intracerebral microdialysis to monitor the hourly concentrations of glutamate in the seizure onset area (epileptogenic hippocampus) over several days. We observed significant 24-h oscillations of extracellular glutamate in the epileptogenic hippocampus (n = 4, JTK_CYCLE test, p < 0.05), but not in the hippocampus of control animals (n = 4). To our knowledge, circadian glutamate oscillations have not been observed in a seizure onset region, and we speculate that the oscillations contribute to the rhythmicity of seizures in MTLE.

(Invited) Conjoint Measurement of Brain Electrophysiology and Neurochemistry

Purpose: We routinely perform conjoint measurement of intracranial electrophysiology and brain neurochemistry in patients with medically intractable epilepsy undergoing intracranial EEG (icEEG) monitoring for possible epilepsy surgery. The conjoint measurement of these two modalities can be revealing of the presumed neurochemical basis of electrophysiological markers of human brain excitation and inhibition. This study focused on interictal electroencephalography (EEG) spikes. Interictal EEG spikes have traditionally been considered to be a marker of brain excitability in epilepsy, though some recent research has questioned this assumption. Here we correlated co-localized measurements of intracranially recorded interictal EEG spikes and basal extracellular glutamate concentrations, a well-established marker of excitation, in patients with intractable epilepsy. Methods: Glutamate was quantified from brain microdialysate obtained from 30 combined depth icEEG electrode and microdialysis catheters placed in 15 patients. EEG spikes were counted, at the time when glutamate was documented, in two separate manners: (1) for the two icEEG electrode contacts on either side of the microdialysis membrane, and (2) for all contacts within the brain region where the dialysis probe was located. Results:We observed the interictal icEEG spike rate was not directly proportional to basal glutamate; rather the icEEG spike rate was low when basal glutamate was high, and vice versa. A joint analysis of brain electrophysiology and neurochemistry thus indicates an inverse, rather than a direct, relationship between interictal icEEG spikes and brain extracellular glutamate. Discussion: Interictal EEG spikes have traditionally been considered to be a marker of brain excitability. The lack of a direct relation between basal glutamate and icEEG spike rate, however, supports the suggestion that EEG spikes may not be reflective of brain excitability, but rather may be reflective of inhibition. To support the findings of this study and underscore the importance of the conjoint measurement of brain electrophysiology and neurochemistry we discuss observations of other studies of interictal EEG spikes, and observations from other studies of conjoint brain electrophysiology and neurochemistry performed by our group. Conclusions: This study suggests that there is considerable value for in-vivo co-localized measurement of human brain electrophysiology and neurochemistry to improve our understanding of these two modalities, and for understanding the mechanism underlying medically intractable epilepsy.

Standard-sampling microdialysis and spreading depolarizations in patients with malignant hemispheric stroke

Spreading depolarizations (SD) occur in high frequency in patients with malignant hemispheric stroke (MHS). Experimentally, SDs cause marked increases in glutamate and lactate, whereas glucose decreases. Here, we studied extracellular brain glutamate, glucose, lactate, pyruvate and the lactate/pyruvate ratio in relationship to SDs after MHS. We inserted two microdialysis probes in peri-infarct tissue at 5 and 15 mm to the infarct in close proximity to a subdural electrode strip. During 2356.6 monitoring hours, electrocorticography (ECoG) revealed 697 SDs in 16 of 18 patients. Ninety-nine SDs in electrically active tissue (spreading depressions, SDd) were single (SDds) and 485 clustered (SDdc), whereas 10 SDs with at least one electrode in electrically inactive tissue (isoelectric SDs, SDi) were single (SDis) and 103 clustered (SDic). More SDs and a significant number of clustered SDs occurred during the first 36 h post-surgery when glutamate was significantly elevated (> 100 µM). In a grouped analysis, we observed minor glutamate elevations with more than two SDs per hour. Glucose slightly decreased during SDic at 5 mm from the infarct. Directions of SD-related metabolic changes correspond to the experimental setting but the long sampling time of standard microdialysis precludes a more adequate account of the dynamics revealed by ECoG.

Clinical aspects of urea cycle dysfunction and altered brain energy metabolism on modulation of glutamate receptors and transporters in acute and chronic hyperammonemia.

In living organisms, nitrogen arise primarily as ammonia (NH3) and ammonium (NH4(+)), which is a main component of the nucleic acid pool and proteins. Although nitrogen is essential for growth and maintenance in animals, but when the nitrogenous compounds exceeds the normal range which can quickly lead to toxicity and death. Urea cycle is the common pathway for the disposal of excess nitrogen through urea biosynthesis. Hyperammonemia is a consistent finding in many neurological disorders including congenital urea cycle disorders, reye's syndrome and acute liver failure leads to deleterious effects. Hyperammonemia and liver failure results in glutamatergic neurotransmission which contributes to the alteration in the function of the glutamate-nitric oxide-cGMP pathway, modulates the important cerebral process. Even though ammonia is essential for normal functioning of the central nervous system (CNS), in particular high concentrations of ammonia exposure to the brain leads to the alterations of glutamate transport by the transporters. Several glutamate transporters have been recognized in the central nervous system and each has a unique physiological property and distribution. The loss of glutamate transporter activity in brain during acute liver failure and hyperammonemia is allied with increased extracellular brain glutamate concentrations which may be conscientious for the cerebral edema and ultimately cell death.

Beyond the "First Hit": Marked Inhibition by N-Acetyl Cysteine of Chronic Ethanol Intake But Not of Early Ethanol Intake. Parallel Effects on Ethanol-Induced Saccharin Motivation.

A number of studies have shown that acetaldehyde synthesized in the brain is necessary to induce ethanol (EtOH) reinforcement in naïve animals (acquisition phase). However, after chronic intake is achieved (maintenance phase), EtOH intake becomes independent of acetaldehyde generation or its levels. Glutamate has been reported to be associated with the maintenance of chronic EtOH intake. The levels of brain extracellular glutamate are modulated by 2 glial processes: glutamate reabsorption via an Na(+) -glutamate transporter (GLT1) and a cystine-glutamate exchanger. Chronic EtOH intake lowers GLT1 levels and increases extracellular glutamate. The administration of N-acetyl cysteine (NAC), a precursor of cystine, has been shown to reduce the relapse of several drugs of abuse, while NAC has not been tested on chronic EtOH intake or on EtOH's influence on the motivation for another drug. These were investigated in the present study. (i) Rats bred for their high EtOH intake were allowed access to 10% EtOH and water up to 87days. NAC was administered (30 and 60mg/kg daily, intraperitoneally) for 14 consecutive days, either during the acquisition phase or the maintenance phase of EtOH drinking. (ii) In additional experiments, rats were allowed EtOH (10%) and water access for 61days, after which EtOH was replaced by saccharin (0.3%) to determine both if chronic EtOH consumption influences saccharin intake and whether NAC modifies the post chronic EtOH saccharin intake. NAC did not influence the acquisition ("first hit") of chronic EtOH intake, but greatly inhibited (60 to 70%; p<0.0001) EtOH intake when NAC was administered to animals that were consuming EtOH chronically. NAC did not influence saccharin intake in naïve animals. In animals that had consumed EtOH chronically and were thereafter offered a saccharin solution (0.3%), saccharin intake increased over 100% versus that of EtOH-untreated animals, an effect that was fully suppressed by NAC. N-acetyl cysteine, a drug approved for use in humans, markedly reduces chronic EtOH intake and abolishes the increased intake of saccharin stimulated by chronic EtOH drinking.

Cerebral Taurine Levels are Associated with Brain Edema and Delayed Cerebral Infarction in Patients with Aneurysmal Subarachnoid Hemorrhage.

Cerebral edema and delayed cerebral infarction (DCI) are common complications after aneurysmal subarachnoid hemorrhage (aSAH) and associated with poor functional outcome. Experimental data suggest that the amino acid taurine is released into the brain extracellular space secondary to cytotoxic edema and brain tissue hypoxia, and therefore may serve as a biomarker for secondary brain injury after aSAH. On the other hand, neuroprotective mechanisms of taurine treatment have been described in the experimental setting. We analyzed cerebral taurine levels using high-performance liquid chromatography in the brain extracellular fluid of 25 consecutive aSAH patients with multimodal neuromonitoring including cerebral microdialysis (CMD). Patient characteristics and clinical course were prospectively recorded. Associations with CMD-taurine levels were analyzed using generalized estimating equations with an autoregressive process to handle repeated observations within subjects. CMD-taurine levels were highest in the first days after aSAH (11.2±3.2µM/l) and significantly decreased over time (p<0.001). Patients with brain edema on admission or during hospitalization (N=20; 80%) and patients developing DCI (N=5; 20%) had higher brain extracellular taurine levels compared to those without (Wald=7.3, df=1, p<0.01; Wald=10.1, df=1, p=0.001, respectively) even after adjusting for disease severity and CMD-probe location. There was no correlation between parenteral taurine supplementation and brain extracellular taurine (p=0.6). Moreover, a significant correlation with brain extracellular glutamate (r=0.82, p<0.001), lactate (r=0.56, p<0.02), pyruvate (r=0.39, p<0.01), potassium (r=0.37, p=0.01), and lactate-to-pyruvate ratio (r=0.24, p=0.02) was found. Significantly higher CMD-taurine levels were found in patients with brain edema or DCI after aneurysmal subarachnoid hemorrhage. Its value as a potential biomarker deserves further investigation.

Alleviation of glutamate mediated neuronal insult by piroxicam in rodent model of focal cerebral ischemia: a possible mechanism of GABA agonism.

Neurotransmitter imbalance is an inevitable outcome in cerebral ischemia that leads to neuronal death. In the present study, we evaluated the effects of piroxicam, a nonsteroidal anti-inflammatory drug (NSAID), on extracellular brain glutamate and γ-aminobutyric acid (GABA) release, survival time, and neuronal cell death. Transient focal cerebral ischemia in male Charles Foster rat led to neuronal infarction and compromised intrinsic antioxidant status. Thirty-minute preadministration of piroxicam (10 mg/kg b.w.) showed a significant (P < 0.01) reduction in cerebral infarct volume and potentiation of the intrinsic antioxidant status. High-performance liquid chromatography of brain cortex and striatum revealed changes in extracellular concentrations of neurotransmitters which were found to be 0.519 ± 0.44 pmole/mg (GABA); 1.18 ± 0.28 pmole/mg (glutamate), and 0.63 ± 0.21 pmole/mg (serotonin), respectively. Hydroxyl radical (·OH) adduct of salicylate in the frontal cortex and striatum in control, untreated, and treated groups was found to be 0.261 ± 0.06, 0.68 ± 0.52, and 0.401 ± 0.68 pmole/mg, respectively. After stroke, the extracellular level of glutamate in rat brain increases continuously as compared to that of control group. However, piroxicam administration in stroke rat significantly reduced (P < 0.05) elevated extracellular cerebral glutamate. This indicates that piroxicam attenuates extracellular glutamate release and also reduces neuronal cell death due to reduction in oxidative stress in cerebral ischemia. Our results also indicate a consequent increase of extracellular GABA in brain regions administered with piroxicam, which hints that piroxicam alleviates glutamate excitotoxicity possibly by GABA agonism.

P.1.g.097 The effects of some serotonin receptor antagonists on memory processes performance in rats

Glutamate has been implicated in the initiation and spread of seizure activity. Agmatine, an endogenous neuromodulator, is an antagonist of NMDA receptors and has anticonvulsive effects. Whether agmatine regulate glutamate release, as measured by in vivo microdialysis, is not known. In this study, we used pentylenetetrazole (PTZ)-induced seizure model to determine the effect of agmatine on extracellular glutamate in rat brain. We also determined the time course and the amount of agmatine that reached brain after peripheral injection. After i.p. injection of agmatine (50 mg/kg), increase of agmatine in rat cortex and hippocampus was observed in 15 min with levels returning to baseline in one hour. Rats, naïve and implanted with microdialysis cannula into the cortex, were administered PTZ (60 mg/kg, i.p.) with prior injection of agmatine (100 mg/kg, i.p.) or saline. Seizure grades were recorded and microdialysis samples were collected every 15 min for 75 min. Agmatine pre-treatment significantly reduced the seizure grade and increased the onset time. The levels of extracellular glutamate in frontal cortex rose two- to three-fold after PTZ injection and agmatine significantly inhibited this increase. In conclusion, the present data suggest that the anticonvulsant activity of agmatine, in part, could be related to the inhibition glutamate release.

Neuroinflammation and Comorbidity of Pain and Depression

Comorbid depression and chronic pain are highly prevalent in individuals suffering from physical illness. Here, we critically examine the possibility that inflammation is the common mediator of this comorbidity, and we explore the implications of this hypothesis. Inflammation signals the brain to induce sickness responses that include increased pain and negative affect. This is a typical and adaptive response to acute inflammation. However, chronic inflammation induces a transition from these typical sickness behaviors into depression and chronic pain. Several mechanisms can account for the high comorbidity of pain and depression that stem from the precipitating inflammation in physically ill patients. These mechanisms include direct effects of cytokines on the neuronal environment or indirect effects via downregulation of G protein-coupled receptor kinase 2, activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase that generates neurotropic kynurenine metabolites, increased brain extracellular glutamate, and the switch of GABAergic neurotransmission from inhibition to excitation. Despite the existence of many neuroimmune candidate mechanisms for the co-occurrence of depression and chronic pain, little work has been devoted so far to critically assess their mediating role in these comorbid symptoms. Understanding neuroimmune mechanisms that underlie depression and pain comorbidity may yield effective pharmaceutical targets that can treat both conditions simultaneously beyond traditional antidepressants and analgesics.

GPR35 Activation Reduces Ca2+ Transients and Contributes to the Kynurenic Acid-Dependent Reduction of Synaptic Activity at CA3-CA1 Synapses

Limited information is available on the brain expression and role of GPR35, a Gi/o coupled receptor activated by kynurenic acid (KYNA). In mouse cultured astrocytes, we detected GPR35 transcript using RT-PCR and we found that KYNA (0.1 to 100 µM) decreased forskolin (FRSK)-induced cAMP production (p<0.05). Both CID2745687 (3 µM, CID), a recently described GPR35 antagonist, and GPR35 gene silencing significantly prevented the action of KYNA on FRSK-induced cAMP production. In these cultures, we then evaluated whether GPR35 activation was able to modulate intracellular Ca2+ concentration ([Ca2+]i ) and [Ca2+]i fluxes. We found that both KYNA and zaprinast, a phosphodiesterase (PDE) inhibitor and GPR35 agonist, did not modify either basal or peaks of [Ca2+]i induced by challenging the cells with ATP (30 µM). However, the [Ca2+]i plateau phase following peak was significantly attenuated by these compounds in a store-operated Ca2+ channel (SOC)-independent manner. The activation of GPR35 by KYNA and zaprinast was also studied at the CA3-CA1 synapse in the rat hippocampus. Evoked excitatory post synaptic currents (eEPSCs) were recorded from CA1 pyramidal neurons in acute brain slices. The action of KYNA on GPR35 was pharmacologically isolated by using NMDA and α7 nicotinic receptor blockers and resulted in a significant reduction of eEPSC amplitude. This effect was prevented in the presence of CID. Moreover, zaprinast reduced eEPSC amplitude in a PDE5- and cGMP-independent mechanism, thus suggesting that glutamatergic transmission in this area is modulated by GPR35. In conclusion, GPR35 is expressed in cultured astrocytes and its activation modulates cAMP production and [Ca2+]i. GPR35 activation may contribute to KYNA effects on the previously reported decrease of brain extracellular glutamate levels and reduction of excitatory transmission.

Effects of Vitamin B6 on the Brain Glutamate Pyrovate Transaminase and Glutamate Oxaloacetate Transaminase in Young and Old Rats

Excessive amounts of extracellular glutamate in brain are excitotoxic and lead to neuronal death. Glutamate pyrovate transamiase (GPT) and glutamate oxaloacetate transaminase (GOT) catalyze transamination of glutamate to alpha ketoglutarate. Because vitamin B6 is essential for the enzymes activities, this study was undertaken to examine the effect of vitamin B6 on age related changes of this enzyme in rat brain. Three and 30 months old male rats were injected with vitamin B6 and the animal's brains were homogenized in phosphate buffer and the enzymes activities were measured in the super- natant. The activities of the enzymes in aged rats were significantly lower as compared to that of young animals. Vitamin B6 induced activation of the brain enzymes in both ages, however, the activation was significantly pronounced in aged animals. Significant activation of GPT and GOT by vitamin B6 in aged rat brain may be resulted from either lower availability of vitamin B6 in aged animals, or lower affinity of the enzymes for pyridoxal 5-phosphate, which is likely to be related to conformational changes during aging. It is suggested that vitamin B6 may restore the activity of these enzymes during the brain aging.

Evidence for astrocytes as a potential source of the glutamate excess in temporal lobe epilepsy

Increased extracellular brain glutamate has been implicated in the pathophysiology of human refractory temporal lobe epilepsy (TLE), but the cause of the excessive glutamate is unknown. Prior studies by us and others have shown that the glutamate degrading enzyme glutamine synthetase (GS) is deficient in astrocytes in the epileptogenic hippocampal formation in a subset of patients with TLE. We have postulated that the loss of GS in TLE leads to increased glutamate in astrocytes with elevated concentrations of extracellular glutamate and recurrent seizures as the ultimate end-points. Here we test the hypothesis that the deficiency in GS leads to increased glutamate in astrocytes. Rats were chronically infused with methionine sulfoximine (MSO, n=4) into the hippocampal formation to induce GS deficiency and recurrent seizures. A separate group of rats was infused with 0.9% NaCl (saline) as a control (n=6). At least 10days after the start of infusion, once recurrent seizures were established in the MSO-treated rats, the concentration of glutamate was assessed in CA1 of the hippocampal formation by immunogold electron microscopy. The concentration of glutamate was 47% higher in astrocytes in the MSO-treated vs. saline-treated rats (p=0.02), and the ratio of glutamate in astrocytes relative to axon terminals was increased by 74% in the MSO-treated rats (p=0.003). These data support our hypothesis that a deficiency in GS leads to increased glutamate in astrocytes. We additionally propose that the GS-deficient astrocytes in the hippocampal formation in TLE lead to elevated extracellular brain glutamate either through decreased clearance of extracellular glutamate or excessive release of glutamate into the extracellular space from these cells, or a combination of the two.

Evaluation of permselective membranes for optimization of intracerebral amperometric glutamate biosensors

Monitoring of extracellular brain glutamate concentrations by intracerebral biosensors is a promising approach to further investigate the role of this important neurotransmitter. However, amperometric biosensors are typically hampered by Faradaic interference caused by the presence of other electroactive species in the brain, such as ascorbic acid, dopamine, and uric acid. Various permselective membranes are often used on biosensors to prevent this. In this study we evaluated the most commonly used membranes, i.e. nafion, polyphenylenediamine, polypyrrole, polyaniline, and polynaphthol using a novel silica-based platinum electrode.First we selected the membranes with the highest sensitivity for hydrogen peroxide in vitro and an optimal selectivity against electrochemical interferents. Then we evaluated the performances of these membranes in a short lasting (3–4h) in vivo experiment. We found that best in vitro performance was accomplished with biosensors that were protected by a poly(m-phenylenediamine) membrane deposited onto the platinum electrode by cyclic voltammetry. However, post-implantation evaluation of these membranes showed poor selectivity against dopamine. Combination with a previously applied nafion layer did not protect the sensors against acute biofouling; indeed it was even counter effective.Finally, we investigated the ability of our biosensors to monitor the effect of glutamate transport blocker DL-TBOA on modulating glutamate concentrations in the prefrontal cortex of anaesthetized rats. The optimized biosensors recorded a rapid 35-fold increase in extracellular glutamate, and are considered suitable for further exploration in vivo

Genetic variation influences glutamate concentrations in brains of patients with multiple sclerosis

Glutamate is the main excitatory neurotransmitter in the mammalian brain. Appropriate transmission of nerve impulses through glutamatergic synapses is required throughout the brain and forms the basis of many processes including learning and memory. However, abnormally high levels of extracellular brain glutamate can lead to neuroaxonal cell death. We have previously reported elevated glutamate levels in the brains of patients suffering from multiple sclerosis. Here two complementary analyses to assess the extent of genomic control over glutamate levels were used. First, a genome-wide association analysis in 382 patients with multiple sclerosis using brain glutamate concentration as a quantitative trait was conducted. In a second approach, a protein interaction network was used to find associated genes within the same pathway. The top associated marker was rs794185 (P < 6.44 x 10(-7)), a non-coding single nucleotide polymorphism within the gene sulphatase modifying factor 1. Our pathway approach identified a module composed of 70 genes with high relevance to glutamate biology. Individuals carrying a higher number of associated alleles from genes in this module showed the highest levels of glutamate. These individuals also showed greater decreases in N-acetylaspartate and in brain volume over 1 year of follow-up. Patients were then stratified by the amount of annual brain volume loss and the same approach was performed in the 'high' (n = 250) and 'low' (n = 132) neurodegeneration groups. The association with rs794185 was highly significant in the group with high neurodegeneration. Further, results from the network-based pathway analysis remained largely unchanged even after stratification. Results from these analyses indicated that variance in the activity of neurochemical pathways implicated in neurodegeneration is explained, at least in part, by the inheritance of common genetic polymorphisms. Spectroscopy-based imaging provides a novel quantitative endophenotype for genetic association studies directed towards identifying new factors that contribute to the heterogeneity of clinical expression of multiple sclerosis.

Altered glial–neuronal crosstalk: Cornerstone in the pathogenesis of hepatic encephalopathy

Hepatic encephalopathy (HE) is a serious neuropsychiatric complication of liver failure, characterized neuropathologically by astrocyte swelling, microglial activation and Alzheimer Type II astrocytosis. Molecular studies in HE brain reveal altered expression of genes coding for key astroglial proteins including early losses of expression of GFAP and the glutamate transporter EEAT-2 with concomitant increases of the astrocytic/microglial mitochondrial benzodiazepine receptor (MBR). Decreased expression of EAAT-2 results in decreased glutamate transport and impaired cycling of glutamate–glutamine between astrocytes and neurons, as well as increased extracellular glutamate, activation of the NMDA receptor-mediated cGMP-NO signal transduction pathway, and nitration of tyrosine residues on key astroglial proteins such as glutamine synthetase (GS) and the MBR. GS is uniquely responsible for the removal of excess ammonia in brain. Ammonia-induced activation of MBR in astrocytes and/or microglia results in stimulation of the synthesis of neurosteroids such as allopregnanolone with positive allosteric GABA-A receptor neuromodulatory properties. Allopregnanolone concentrations are increased up to 7-fold in HE brain. Attenuation of microglial activation by minocycline results in a delay in onset of HE and prevents brain edema in liver failure. Mild hypothermia is likewise beneficial in acute liver failure resulting in normalization of extracellular brain glutamate and prevention of oxidative/nitrosative stress in experimental animals with HE resulting from either ischemic or toxic liver injuries.

Change in Brain Glucose After Enteral Nutrition in Subarachnoid Hemorrhage

The alteration of brain extracellular glucose after enteral nutrition (EN) remains unclear. In this study, we used brain microdialysis methods to estimate whether the physiologic elevation of plasma glucose following EN affects brain glucose metabolism of aneurysmal subarachnoid hemorrhage (SAH) patients. Brain extracellular glucose, lactate, glycerol, glutamate, and pyruvate were measured with a brain microdialysis probe in 12 patients (mean age: 60.0 y+/-7.8 y) after SAH. The EN was initially administered a mean of 3.2 d after the onset of SAH. All of the measured parameters were estimated before and after EN. Cerebral perfusion pressure did not significantly change after SAH during the study period. Plasma glucose rose significantly after EN (141.4+/-11.6mg/dL before EN versus 183.8+/-26.2mg/dL immediately after EN (P=0.0006), 177.7+/-30.2mg/dL at 2h after EN (P=0.0033)). The brain extracellular glucose before EN (2.5+/-0.92mmol/L) was significantly lower than the levels measured just after (3.49+/-1.0mmol/L, P=0.0186) and 2h after the end of EN (3.70+/-1.0mmol/L, P=0.0053). Brain extracellular concentrations of lactate, glutamate, pyruvate, and glycerol showed no significant changes. Brain extracellular glucose increased after the transient elevation of plasma glucose following EN. These results suggest that brief, physiologic elevations in plasma glucose after EN produced no changes in brain extracellular glutamate concentration or lactate/pyruvate ratio. These data may help determine the plasma glucose levels most effective for avoiding brain metabolic acidosis in patients after SAH. It remains unclear, however, how SAH itself influences these findings.

Simvastatin Reduces the Association of NMDA Receptors to Lipid Rafts

Excess brain extracellular glutamate induced by cerebral ischemia leads to neuronal death, mainly through overactivation of N-methyl-D-aspartate (NMDA) receptors. The cholesterol-lowering drugs statins have been reported to protect from NMDA-induced neuronal death but, so far, the mechanism underlying this protection remains unclear. Because NMDA receptors have been reported to be associated with the cholesterol-rich membrane domains known as lipid rafts, we have investigated the effect of treatments that deplete cholesterol levels on excitotoxicity and on association of NMDA receptors to lipid rafts. Primary neuronal cultures were pretreated with inhibitors of cholesterol synthesis and cholesterol, and NMDA-induced cell death was determined by measuring release of lactate dehydrogenase. Lipid raft fractions were isolated and Western blots were performed. Treatment with the inhibitors of cholesterol synthesis simvastatin, which inhibits the first step of cholesterol synthesis, or AY9944, which inhibits the last step of cholesterol synthesis, protected neurons from NMDA-induced neuronal death by 70% and 54%, respectively. Treatment with these compounds reduced neuronal cholesterol levels by 35% and 13%, respectively. Simvastatin and AY9944 reduced the association of the subunit 1 of NMDA receptors (NMDAR1) to lipid rafts by 42% and 21%, respectively, and did not change total expression of NMDAR1. Addition of cholesterol reduced neuroprotection by statins and AY9944, and partially reverted the effect of simvastatin on the association of NMDAR1 to lipid rafts. These data demonstrate that reduction of cholesterol levels protects from NMDA-induced neuronal damage probably by reducing the association of NMDA receptors to lipid rafts.