Anti-GluA3 antibodies in frontotemporal dementia: effects on glutamatergic neurotransmission and synaptic failure.

Despite the great effort of the scientific community in the field, the pathogenesis of frontotemporal dementia (FTD) remains elusive. Recently, a role for autoimmunity and altered glutamatergic neurotransmission in triggering disease onset has been put forward. We reported the presence of autoantibodies recognizing the GluA3 subunit of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in about 25% of FTD cases. In this study, we evaluated the mechanisms involved in anti-GluA3 autoimmunity, through molecular/neurochemical analyses conducted on patients' brain specimens with frontotemporal lobar degeneration-tau neuropathology. We then corroborated these results in vivo in FTD patients with transcranial magnetic stimulation and glutamate, D-serine, and L-serine dosages in the cerebrospinal fluid and serum. We observed that GluA3 autoantibodies affect glutamatergic neurotransmission, decreasing glutamate release and altering GluA3-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor levels. These alterations were accompanied by changes of scaffolding proteins involved in receptor synaptic retention/internalization. The above results were confirmed by transcranial magnetic stimulation, suggesting a significant impairment of indirect measures of glutamatergic neurotransmission in FTD patients compared with controls, with further add-on harmful effect in those FTD patients with anti-GluA3 antibodies. Finally, FTD patients showed a significant increase of glutamate, D-serine, and L-serine levels in the cerebrospinal fluid.

Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders.

HIV-associated neurocognitive disorders (HAND) have been linked to dysregulation of glutamate metabolism in the central nervous system (CNS) culminating in elevated extracellular glutamate and disrupted glutamatergic neurotransmission. Increased glutamate synthesis via upregulation of glutaminase (GLS) activity in brain immune cells has been identified as one potential source of excess glutamate in HAND. However, direct evidence for this hypothesis in an animal model is lacking, and the viability of GLS as a drug target has not been explored. In this brief report, we demonstrate that GLS inhibition with the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) can reverse cognitive impairment in the EcoHIV-infected mouse model of HAND. However, due to peripheral toxicity DON is not amenable to clinical use in a chronic disease such as HAND. We thus tested JHU083, a novel, brain penetrant DON prodrug predicted to exhibit improved tolerability. Systemic administration of JHU083 reversed cognitive impairment in EcoHIV-infected mice similarly to DON, and simultaneously normalized EcoHIV-induced increases in cerebrospinal fluid (CSF) glutamate and GLS activity in microglia-enriched brain CD11b + cells without observed toxicity. These studies support the mechanistic involvement of elevated microglial GLS activity in HAND pathogenesis, and identify JHU083 as a potential treatment option. Graphical Abstract Please provide Graphical Abstract caption.Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders .

Pharmacokinetics and efficacy of pemetrexed in patients with brain or leptomeningeal metastases.

Brain metastases (BM) and leptomeningeal metastases (LM) are devastating neurologic complications. Pemetrexed is a multi-targeted anti-folate agent approved for treatment of nonsquamous non-small cell lung cancer but has anti-tumor activity in other solid tumors. We performed two trials using pemetrexed in patients with BM and LM to assess CSF penetration and anti-tumor activity. Patients were treated with intravenous pemetrexed at doses of 500 (n = 3), 750 (n = 3), 900 (n = 12) or 1,050 mg/m(2) (n = 3) every 3 weeks. Neuro-imaging was done every 6 weeks. Matched CSF and plasma samples were obtained serially from three patients with Ommaya reservoirs; the remaining patients had a single paired collection. Twenty-one patients (15 women and six men) with median age of 50 years and median KPS of 90 were treated. Primary tumors included breast (13), lung (4), colorectal (1), endometrial (1), esophageal (1) and pinealoblastoma (1). Nine patients had prior whole brain RT and median number of prior chemotherapies was two including prior methotrexate in four patients. Median pemetrexed doses administered was three (range 1-14). Responses included one partial response, ten stable disease and ten progressive disease. Median time to progression and survival was 2.7 and 7.3 months; PFS six was 22 %. No major toxicities were seen. Pemetrexed distributed from the plasma to the CSF within 1-4 h with the resulting CSF concentrations < 5 % of plasma. Pemetrexed was tolerated in solid tumor patients with CNS metastases. Limited anti-tumor activity was seen, which might have been due to low CSF concentrations, although some patients displayed prolonged benefit.

Acute depression of energy metabolism after microdialysis probe implantation is distinct from ischemia-induced changes in mouse brain.

The current study used measurements of metabolites and markers of membrane integrity to determine the most suitable time point for microdialysis experiments following probe implantation. Leakage of Evans blue and sodium fluorescein indicated increased BBB permeability only immediately (15 min), but not 1.5 and 24 h following probe implantation. Acute implantation decreased glucose and lactate levels relative to the levels after 24 h (to 13-37% and 25-60%, respectively). No change in extracellular levels of glutamate or glycerol was seen. In comparison to acute probe implantation, the pattern of damage under brain ischemia (middle cerebral artery occlusion) differed: While glucose levels dropped, lactate levels rose after ischemia, and glutamate (tenfold) and glycerol (eightfold) increased sharply. In conclusion, acute implantation of a microdialysis probe causes transient depression of the energy metabolites, glucose and lactate, likely due to injury-induced hypermetabolism. However, no massive tissue damage or severe ischemic conditions around the probe occur.

In vivo effects of antibodies from patients with anti-NMDA receptor encephalitis: further evidence of synaptic glutamatergic dysfunction.

BACKGROUND: A severe encephalitis that associates with auto-antibodies to the NR1 subunit of the NMDA receptor (NMDA-R) was recently reported. Patients' antibodies cause a decrease of the density of NMDA-R and synaptic mediated currents, but the in vivo effects on the extracellular glutamate and glutamatergic transmission are unknown. METHODS: We investigated the acute metabolic effects of patients' CSF and purified IgG injected in vivo. Injections were performed in CA1 area of Ammon's horn and in premotor cortex in rats. RESULTS: Patient's CSF increased the concentrations of glutamate in the extracellular space. The increase was dose-dependent and was dramatic with purified IgG. Patients' CSF impaired both the NMDA- and the AMPA-mediated synaptic regulation of glutamate, and did not affect the glial transport of glutamate. Blockade of GABA-A receptors was associated with a marked elevation of extra-cellular levels of glutamate following a pretreatment with patients' CSF. CONCLUSION: These results support a direct role of NMDA-R antibodies upon altering glutamatergic transmission. Furthermore, we provide additional evidence in vivo that NMDA-R antibodies deregulate the glutamatergic pathways and that the encephalitis associated with these antibodies is an auto-immune synaptic disorder.

Glutamate export at the choroid plexus in health, thiamin deficiency, and ethanol intoxication: review and hypothesis.

INTRODUCTION: The earliest observed effect in the pathogenesis of experimental Wernicke's encephalopathy and of ethanol intoxication in rats is impairment of the blood cerebrospinal fluid (CSF) barrier at the choroid plexus (CP). For an explanation, these observations direct attention to the role of the CP in maintaining glutamate homeostasis in the CSF. METHODS: Characteristics of the CP epithelium (CPE) are reviewed, focusing on its role in removal of glutamate from the CSF and its potential for impairment by ethanol oxidation or by thiamin-deficient glucose oxidation. RESULTS: The export of glutamate from CSF to blood at the CP is energy dependent, saturable, and stereospecific. However, the incapacity of the CP to convert glutamate to other metabolites makes it vulnerable to glutamate accumulation should alpha-ketoglutarate dehydrogenase activity be decreased. Elsewhere ethanol metabolism and thiamin-deficiency independently decrease the activity of this mitochondrial enzyme. We argue that they have the same effect within the mitochondria-rich CPE, thereby decreasing energy production necessary for export of glutamate from CSF to blood; diverting its energy metabolism to further glutamate production; and impairing its blood CSF barrier function. This impairment appears to be mediated by glutamate and is attenuated by MK801 but whether it involves one of the CPE glutamate receptors is yet uncertain. This impairment exposes the CSF and hence the paraventricular brain extracellular fluid to neuroactive substances from the blood, including further glutamate, explaining the paraventricular location of neuropathology in Wernicke's encephalopathy. Other organs normally protected from blood by a barrier are affected also by ethanol abuse and by thiamin deficiency, namely the eye, peripheral nerves, and the testis. Much less is known regarding the function of these barriers. CONCLUSIONS: Impairment of the CP by ethanol intoxication and by thiamin-deficient carbohydrate metabolism has a common, rational explanation that can guide future research.

Peripheral markers of glutamatergic dysfunction in neurological diseases: focus on ex vivo tools.

Since the proposal that excessive glutamatergic stimulation could be responsible for neuronal suffering and death, excitotoxicity and glutamate uptake deficits have been repeatedly confirmed to play a key role in the pathogenesis of different neurological diseases. Therefore, it is conceivable that assessing the glutamatergic system function directly in patients could be extremely useful for early diagnosis, prognostic evaluation, and optimization of the therapy. A possibility is offered by assessing glutamate levels in biological fluid, such as plasma and CSF, where increased levels of this amino acid have been reported in patients affected by stroke, amyotrophic lateral sclerosis (ALS), and AIDS dementia complex. However, the metabolic role of this amino acid acts as a confounding factor, and the possibility of directly assessing glutamatergic functional parameters, such as amino acid reuptake, would probably mirror closely the actual excitotoxic damage operative in each patient. Here we will describe our findings obtained in peripheral ex vivo cells, such as platelets and fibroblasts, both displaying a functional glutamate reuptake system. Consistent with a systemic-impairment assumption, glutamate uptake was shown to be reduced in peripheral cells of Alzheimer's disease, Down syndrome, Parkinson's disease, ALS, and stroke patients. Different systemic factors might be responsible for this phenomenon, including genetic predisposition, oxidative stress, and inflammatory response, raising new, exciting questions about the relevance of their possible interactions for the pathogenesis of neurological disorders.

Elevated cerebrospinal fluid levels of glutamate in children with bacterial meningitis as a predictor of the development of seizures or other adverse outcomes.

OBJECTIVE: Evaluation of elevated cerebrospinal fluid levels of glutamate in children with bacterial meningitis as a predictor of seizures or other adverse outcomes. DESIGN: Prospective cohort study with controls. SETTING: A 36-bed pediatric intensive care unit and primary pediatric referral center. PATIENTS: From 1999 to 2001, a total of 55 patients, between the ages of 0 and 18 yrs, with lumbar punctures performed for suspected meningitis. MEASUREMENTS AND MAIN RESULTS: A total of 23 patients had bacterial meningitis confirmed by cerebrospinal fluid/blood culture and elevated cerebrospinal fluid white blood cell counts, and 32 patients, who tested negative, were included as controls. The median age for the patients with meningitis was 1.0 yr (range, 0.0-15.2 yrs), and in the culture-negative group (control group), the median age was 0.3 yrs (range, 0.0-17.0 yrs). The average cerebrospinal fluid white blood cell count was 2707 +/- 3897 in the group with bacterial infection, whereas in the control group, the average was 148 +/- 259 (p < .01). Patients with bacterial meningitis had a mean cerebrospinal fluid glutamate level of 60.5 +/- 88.4 mol/L, whereas the mean cerebrospinal fluid glutamate level in the control group was 4.9 +/- 11.0 mol/L (p < .01). However, only 10 of 23 children with bacterial meningitis had a second lumbar puncture performed during the study. There was no correlation between the cerebrospinal fluid white blood cell count and cerebrospinal fluid glutamate levels in either the study or control patients. None of the control patients developed seizures or neurologic deficits, despite some patients having elevated glutamate levels. However, four patients with bacterial meningitis developed seizures after admission to the hospital, and ten were discharged with at least some neurologic sequelae attributable to their infection. Two out of the three who developed seizures and had a repeat lumbar puncture demonstrated persistent elevation of cerebrospinal fluid glutamate levels. In addition, 70% of patients (7 of 10) with Streptococcus pneumoniae meningitis developed neurologic complications (p = .04). CONCLUSIONS: Bacterial meningitis in children causes an increase in cerebrospinal fluid glutamate that in many cases persists over time. However, in this limited study, neither higher nor persistent elevation of cerebrospinal fluid glutamate levels is predictive of which patients might develop seizures or other apparent immediate adverse outcomes after invasive infection. The responsible organism seems to have far more significance in predicting the development of adverse sequelae.

Headache as a surrogate marker of the molecular mechanisms implicated in progressing stroke.

The mechanism for headache in patients with acute ischaemic stroke are not completely understood. We analysed the relationship between headache and the early worsening of neurological symptoms in patients with acute ischaemic stroke, and we studied the possible biochemical mechanisms implicated. Headache at the onset of ischaemic stroke predicted progression with a sensitivity, specificity, and positive predictive value of 56%, 99%, and 98%, respectively. CSF concentrations of glutamate, Interleukin-6, and NO-m were significantly greater in patients with progressing stroke than in patients with nonprogressing stroke, and these biochemical markers were also significantly higher in patients with headache than in those without headache. Results of this study suggest that headache at the onset of ischaemic stroke is an independent predictor of neurological worsening and we hypothesize that headache might be a surrogate marker of the molecular mechanisms involved in neurological worsening after acute stroke.

Increased cerebrospinal fluid and serum nitrite and nitrate levels in amyotrophic lateral sclerosis.

Abnormal glutamate metabolism is implied in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and cerebrospinal fluid (CSF) glutamate levels appear to be elevated. Since nitric oxide (NO) inhibits glutamate transport, excessive amounts of nitric oxide could underlie the glutamate induced neurotoxicity in ALS. Stable metabolites of NO (NO2- + NO3-) levels were determined in serum and CSF of sporadic ALS patients and control subjects. NO2- + NO3- levels were higher in ALS, in males and in serum samples compared to controls, females and CSF, respectively. Furthermore, while the difference between serum and CSF NO2- + NO3- levels was significant in males (higher in serum) no such difference was observed in females. Our results suggest that nitric oxide may be involved in the pathogenesis of ALS directly or indirectly and in a sexually dimorphic manner.

Gender effects on persistent cerebral metabolite changes in the frontal lobes of abstinent cocaine users.

OBJECTIVE: Previous studies found functional changes in the frontal brain region and regions with projections to the frontal lobe in cocaine users. The aim of this study was to investigate persistent neurochemical changes in the frontal lobes of subjects with a history of crack cocaine dependence and to determine whether these changes are different in male and female users. METHOD: The frontal gray and white matter of 64 young asymptomatic and abstinent (> 5 months) cocaine users (34 male and 30 female) and 58 healthy comparison subjects without a history of drug abuse was evaluated with localized proton magnetic resonance spectroscopy (1H-MRS). RESULTS: Two-way analysis of variance showed significant cocaine effects on the concentration of frontal gray matter N-acetyl compounds, on the ratio of frontal white matter N-acetyl compounds to creatine levels, on frontal gray and white matter myoinositol levels, and on the ratio of myoinositol to creatine. Significant gender effects were observed for frontal gray matter choline-containing compounds, the ratio of choline-containing compounds to creatine, and the percentage of CSF in both gray and white matter. Interaction effects of cocaine and gender were observed for creatine, N-acetyl/creatine ratio, and myoinositol/creatine ratio in frontal white matter. CONCLUSIONS: Cocaine use is associated with neuronal injury (with decreased N-acetyl compounds) in the frontal cortex and glial activation (with increased myoinositol) in both frontal gray and white matter. In the frontal lobe, cocaine affects male users differently than female users. Future studies on the effects of cocaine abuse should control for the effects of gender-specific neurotoxicity.

Markers of glutamatergic neurotransmission and oxidative stress associated with tardive dyskinesia.

OBJECTIVE: Tardive dyskinesia is a movement disorder affecting 20%-40% of patients treated chronically with neuroleptic drugs. The dopamine supersensitivity hypothesis cannot account for the time course of tardive dyskinesia or for the persistence of tardive dyskinesia and the associated structural changes after neuroleptics are discontinued. The authors hypothesized that neuroleptics enhance striatal glutamatergic neurotransmission by blocking presynaptic dopamine receptors, which causes neuronal damage as a consequence of oxidative stress. METHOD: CSF was obtained from 20 patients with schizophrenia, 11 of whom had tardive dyskinesia. Markers for oxidative stress, including superoxide dismutase, lipid hydroperoxide, and protein carbonyl groups, and markers for excitatory neurotransmission, including N-acetylaspartate, N-acetylaspartylglutamate, aspartate, and glutamate, were measured in the CSF specimens. Patients were also rated for tardive dyskinesia symptoms with the Abnormal Involuntary Movement Scale. RESULTS: Tardive dyskinesia patients had significantly higher concentrations of N-acetylaspartate, N-acetylaspartylglutamate, and aspartate in their CSF than patients without tardive dyskinesia when age and neuroleptic dose were controlled for. The significance of the higher levels of protein-oxidized products associated with tardive dyskinesia did not pass Bonferroni correction, however. Tardive dyskinesia symptoms correlated positively with markers of excitatory neurotransmission and protein carbonyl group and negatively with CSF superoxide dismutase activity. CONCLUSIONS: These findings suggest that there are elevated levels of oxidative stress and glutamatergic neurotransmission in tardive dyskinesia, both of which may be relevant to the pathophysiology of tardive dyskinesia.

Excitatory amino acids in cerebrospinal fluid and their relations with clinical features and outcomes in acute head injury.

OBJECTIVE: To determine the contents and dynamics of excitatory amino acids (EAAs), glutamate (GLU) and aspartate (ASP) in the cerebrospinal fluid (CSF) of patients with acute head injury and to clarify the relationship of EAAs with clinical features and outcomes. METHODS: Forty-two adults with acute head injury were included. Glasgow coma scale (GCS) obtained at admission and Glasgow outcome scale (GOS) obtained three months post-injury were assessed. Samples of CSF were obtained via ventricular or lumbar puncture every 24 hours. GLU and ASP in CSF were analyzed by reversed-phase HPLC with a fluorescent detector. Nine control subjects were adults with lumbar anesthesia but without neurological diseases. RESULTS: The peak concentration of GLU and ASP of head-injured group was significantly higher than that of the control group. Dynamic research on severely injured ones indicated that the peak value of GLU mostly appeared within 48 hours post-injury; it decreased with the improving or remaining of the neurological status, and increased with the deterioration, but was still higher than that of the control seven days post-injury. The peak value of EAAs of severely injured patients was conspicuously higher than that of mildly injured group. There was a significant negative correlation between the peak values of GCS and EAAs. The peak value of EAAs in patients with poor outcome was remarkably higher than that in patients with good outcome. GOS was closely correlated to the peak value of EAAs. When the concentration of GLU was over 7 mumol/L, the rate of poor-outcome increased markedly. CONCLUSION: The content of EAAs in CSF increases following acute head injury and remains higher at least a week post-injury in severely injured patients. The more severe the trauma, the more obvious the excitotoxicity induced by EAAs; the more serious the secondary brain insult and the brain edema will be, the worse the outcome, naturally.

Cerebrospinal fluid gamma-aminobutyric acid and glutamate values in dogs with epilepsy.

OBJECTIVES: To investigate changes in CSF concentrations of inhibitory and excitatory neurotransmitters in dogs with confirmed idiopathic epilepsy, and to evaluate them with regard to the clinical characteristics of the sample population and of the seizures. ANIMALS: 13 (8 male and 5 female) drug-naive dogs with an initial generalized seizure, 6 (4 male and 2 female) drug-naive dogs with an initial partial seizure, and 10 clinically normal (5 male and 5 female) control dogs. PROCEDURE: At least 24 hours after the last observed seizure, CSF was collected aseptically from the cisterna cerebellomedullaris, and a portion was immediately aliquoted into vials, placed on dry ice, then stored at -80 C. The CSF glutamate (GLU) and gamma-aminobutyric acid (GABA) concentrations were estimated by use of HPLC with electrochemical detection. Cerebellomedullary cisternal CSF glutamate and GABA values were analyzed in dogs with noninduced idiopathic epilepsy. Comparisons were determined for differences attributable to weight, sex, age at first seizure, seizure type, and total time of past seizure history. RESULTS: Mean (range) age at onset of the first seizure was 3.33 (0.1 to 11), 3.5 (0.1 to 11), and 3.25 (0.5 to 9) years for all dogs, dogs with initial partial seizure, and dogs with initial generalized seizure, respectively. Low GABA and high GLU values were found in the CSF of epileptic dogs, and were independent of time elapsed between the first observed seizure and CSF sample collection. The GABA value was inversely related to body weight in epileptic dogs, independent of age. Changes in GABA and GLU concentrations were not related to seizure type. CONCLUSIONS: Altered GABA and GLU values in CSF might be indicative of a state of chronic overexcitation in the brain of dogs with primary or idiopathic epilepsy. CLINICAL RELEVANCE: CSF GABA and GLU may serve as important markers in epileptic dogs for potential response to antiepileptic drugs.

Glutamate in pyridoxine-dependent epilepsy: neurotoxic glutamate concentration in the cerebrospinal fluid and its normalization by pyridoxine.

BACKGROUND: Pyridoxine-dependent epilepsy is a rare autosomal recessive disorder. Untreated patients suffer from a progressive encephalopathy with mental retardation, intractable epilepsy, and progressive neurological signs and symptoms. Lifelong supplementation with vitamin B6 is the treatment of choice. However, despite early treatment, many patients develop mental retardation. OBJECTIVES: To assess the role of glutamate as an excitatory neurotransmitter and neurotoxin in pyridoxine-dependent epilepsy. METHODS: We examined cerebrospinal fluid (CSF) levels of glutamate, gamma-aminobutyric acid, and pyridoxal-5'-phosphate in a patient with pyridoxine dependency while on and off vitamin B6 treatment. RESULTS: Off vitamin B6 the glutamate level was two hundred times normal. An intermediate dose of vitamin B6 (5 mg/kg BW/day) caused normalization of the EEG and remission of the seizures, but the CSF glutamate concentration was still ten times normal. With a higher dose of pyridoxine (10 mg/kg BW/day) the CSF glutamic acid normalized. CONCLUSIONS: The results indicate that control of epilepsy might not suffice as the therapeutic aim in treating of pyridoxine dependency. In view of the evidence for the role of excitatory amino acids in destruction of CNS nerve cells, the optimal treatment must counteract the raised levels of CSF glutamate and the dosage of vitamin B6 must be adjusted accordingly. The development of mental retardation might theoretically be prevented by adjusting the dose of vitamin B6 to achieve not only remission of epilepsy but also normalization of CSF glutamate.

Lamotrigine in Rett syndrome.

An elevated CSF glutamate level has recently been reported in Rett syndrome. Because the anticonvulsant effect of Lamotrigine is probably due to inhibition of glutamate release, this drug was given as an add-on drug to 4 girls with Rett syndrome. All patients responded with a seizure reduction of 50% or more and an improved well-being. A controlled study at the early stages of the syndrome could be interesting.

Excitatory amino acids in the cerebrospinal fluid of asphyxiated infants: relationship to hypoxic-ischemic encephalopathy.

Asphyxiated (n = 27) and control infants (n = 25) were subjected to spinal taps. Amino acids were measured with liquid chromatography and the degree of hypoxic-ischemic encephalopathy was determined in each case. In asphyxiated infants, the concentrations of aspartate and glutamate were 286% and 387% (p < or = 0.01 and p < or = 0.05) of the control values, respectively. The cerebrospinal fluid aspartate levels were significantly (p < or = 0.05) higher in the group with severe (3.4 mumol/l) compared with the group with mild hypoxic-ischemic encephalopathy (1.0 mumol/l). Glutamate was also higher in the group with severe (12.3 mumol/l) than in the groups with mild (2.7 mumol/l) or moderate (3.2 mumol/l) hypoxic-ischemic encephalopathy (p < or = 0.05). High concentrations of excitatory amino acids were present in the CSF of asphyxiated infants which may exert excitotoxic effects.

Time-related loss of glutamine from hippocampal slices and concomitant changes in neurotransmitter amino acids.

A dramatic, time-dependent loss of L-glutamine was observed in mouse and rat hippocampal slices equilibrated in normal artificial CSF under static (no-flow) and superfused (constant-flow) conditions. Concomitant with the decline in L-glutamine, there was a significant, but less pronounced, decrease in levels of the neurotransmitter amino acids, gamma-aminobutyric acid, L-aspartate, and L-glutamate. The disappearance of L-glutamine was a result of diffusion from the tissue to the artificial CSF rather than chemical or biochemical transformation. The loss of amino acids from the hippocampal slices was prevented to different degrees by the addition of 0.5 mM exogenous L-glutamine to the artificial CSF. The levels of newly synthesized amino acids were also determined, because they may be more indicative of the neuronal activity than the total tissue levels of amino acids. The effects of perturbations in glutamine (length of the equilibration time and addition of exogenous glutamine) on newly synthesized glutamate were more pronounced under 4-aminopyridine-stimulated than control (unstimulated) conditions. Therefore, a loss of L-glutamine from the hippocampal slices may have neurophysiological effects and warrants further investigation.