Human CSF GABA Concentrations: Revised Dowd for Controls, but Not Decreased in Huntington''s Chorea

gamma-Aminobutyric acid (GABA) concentrations were measured in CSF specimens from two large groups of control subjects, one without neurological or psychiatric disease, and one with a variety of neurological disorders not known to involve altered GABAergic function in brain. CSF GABA was also measured in patients with Huntington's chorea and in patients with other choreiform disorders. GABA was measured in CSF by a modification of the ion exchange-fluorometric method that featured use of a relatively large cation exchange column, and a markedly decreased quantity of sulfosalicylic acid for deproteinization of CSF. Mean BABA concentrations in CSF were 87 and 77 nmol/liter for neurologically normal and abnormal control subjects, 82 nmol/liter for the Huntington's chorea patients, and 105 nmol/liter for patients with other forms of chorea. The mean concentration of homocarnosine was not reduced in CSF of Huntington's chorea patients as compared with controls. Mean CSF GABA concentrations found in control subjects were less than half the lowest control means previously reported. These low values are attributable in part to a reduction in on-column hydrolysis of conjugated forms of GABA in CSF, which can be produced by excessive sulfosalicylic acid, and in part to improved chromatographic resolution of GABA from other unknown o-phthalaldehyde-reactive compounds in CSF. Analysis of free GABA in CSF does not appear useful for diagnosis of suspected Huntington's chorea, nor as a possible predictive test for persons genetically at risk for Huntington's chorea.     

Artifactual Increases in the Concentration of Free GABA in Samples of Human Cerebrospinal Fluid Are Due to Degradation of Homocarnosine

Abstract: Samples of untreated human cerebrospinal fluid (CSF) were kept at room temperature (20±1°C) up to 72 h, and changes in γ-aminobutyric acid (GABA) and homocarnosine contents were measured. The concentration of free GABA increased with time, and concomitantly a similar decrease occurred in the concentration of homocarnosine. Total GABA after hydrolysis (present in human CSF at concentrations 40–100 times that of free GABA) did not change. After 2 h the increase in CSF GABA for seven subjects ranged from 42 to 244 pmol/ml. The rate of increase in CSF GABA was positively correlated with the initial homocarnosine concentration. Approximately 5% per h of the initial homocarnosine content was degraded during the first 7 h at room temperature; thereafter the rate gradually decreased. No free GABA was formed in CSF frozen at −70°C for 10 days. When this CSF was restored to room temperature, the formation of free GABA from homocarnosine occurred at essentially the same rate as that observed in fresh CSF. These results demonstrate that the well-known artifactual increase in GABA concentration of untreated human CSF depends on the concentration of homocarnosine. The rapidity of this increase (up to 2 pmollmlimin) could account for disparities among CSF free GABA concentrations previously reported from normal subjects. It is suggested that measurement of concentrations of total GABA in the CSF would provide a better index of human brain GABA concentration than determination of CSF free GABA.     

Clinical relevance of measuring GABA concentrations in cerebrospinal fluid

Determination of GABA concentrations in human cerebrospinal fluid can be used to assess GABA-ergic activity in the central nervous system. As CSF free GABA concentrations may vary with age, sex, CSF fraction, and collection and storage conditions, careful attention to these factors are necessary to allow interpretation of results. Longitudinal studies to investigate the influence of pharmacological agents on CSF GABA have proven especially useful to define clinical biochemical activity and have been utilized to attribute the anti-epileptic action of vigabatrin, a selective inhibitor of GABA-transaminase, to its effects on brain GABA metabolism.Special issue dedicated to Dr. Sidney Udenfriend.     

Transport of GABA at the Blood-CSF Interface

Abstract: The entry of GABA into cerebrospinal fluid (CSF) was studied in dogs anesthetized with pentobarbital and relaxed with suxamethonium. GABA was administered intravenously as a priming dose and subsequent maintenance infusion to compensate for the rapid elimination of the amino acid. Steady state concentrations of GABA in CSF were reached between 10 and 60 min after injection, the rate of entry tending to decrease with increasing plasma levels. During steady state conditions CSF concentrations showed great interin-dividual differences and varied between 0.03 and 5.1% of those in plasma. Probenecid and sodium valproate considerably enhanced the CSF/plasma concentration ratio of GABA. When GABA was directly injected into the liquor space, probenecid slowed down the elimination of GABA from CSF. The results suggest a transport of GABA into and out of CSF, the outward transport being inhibited by probenecid and sodium valproate.     

Free-GABA levels in the cerebrospinal fluid of patients suffering from several neurological diseases Its potential use for the diagnosis of diseases which course with inflammation and tissular necrosis

Summary Free GABA levels were measured in the cerebrospinal fluid (CSF) of 74 neurological patients suffering from cerebral cysticercosis (n = 9), Parkinson's disease (n = 5), multiple sclerosis (n = 6), epilepsy (n = 24), meningeal tuberculosis (n = 6), viral encephalitis (n = 3), cerebrovascular disease (n = 8) and several kinds of dystonia (n = 5). A statistical significant four-fold elevation in free GABA levels was found in patients with cerebral cysticercosis. A non statistical significant two-fold increase in free GABA levels was also encountered in the CSF of patients affected by cerebrovascular disease and viral encephalitis. No changes in CSF free GABA levels were found in patients suffering from any of the other disorders. It is suggested that free GABA levels may be elevated in the CSF of patients suffering from neurological diseases which course with inflammation and tissular necrosis such as cerebral cysticercosis. Much work is needed however to establishd whether CSF free GABA levels can be used as a diagnostic tool in at least some type of these patients.     

Relationship Between GABA Concentrations in Cerebrospinal Fluid and Seizure Excitability

Abstract: Various studies suggest that alterations in GABAergic function may be connected to epileptic seizures. Low CSF GABA levels have been reported in epilepsy and also febrile convulsions of children. In this study the pentet-razole seizure threshold of dogs was compared with the concentration of GABA in the CSF and blood plasma. A highly significant positive correlation was found between seizure excitability and CSF GABA level, but not between CSF and plasma GABA concentrations.     

Concentrations of GABA and Other Amino Acids in CSF from Torsion Dystonia Patients

Abstract: Free amino acid concentrations were measured by conventional amino acid analysis, and γ-aminobutyric acid (GABA) concentrations were determined, by an ion-exchange fluorometric technique, in CSF specimens from 16 patients with torsion dystonias and in CSF from a large number of control subjects. The mean CSF GABA concentration of the dystonia patients (97 ± 11 nmol/L) did not differ significantly from the means for CSF GABA in two groups of adult control subjects. Mean concentrations of all commonly determined amino compounds were normal in the CSF of torsion dystonia patients, except for ornithine, which was modestly but significantly reduced.     

Reduced brain levels of DHEAS in hepatic coma patients: significance for increased GABAergic tone in hepatic encephalopathy

Increased neurosteroids with allosteric modulatory activity on GABA(A) receptors such as 3α-5α tertrahydroprogesterone; allopregnanolone (ALLO), are candidates to explain the phenomenon of "increased GABAergic tone" in hepatic encephalopathy (HE). However, it is not known how changes of other GABA(A) receptor modulators such as dehydroepiandrosterone sulfate (DHEAS) contribute to altered GABAergic tone in HE. Concentrations of DHEAS were measured by radioimmunoassay in frontal cortex samples obtained at autopsy from 11 cirrhotic patients who died in hepatic coma and from an equal number of controls matched for age, gender, and autopsy delay intervals free from hepatic or neurological diseases. To assess whether reduced brain DHEAS contributes to increased GABAergic tone, in vitro patch clamp recordings in rat prefrontal cortex neurons were performed. A significant reduction of DHEAS (5.81±0.88 ng/g tissue) compared to control values (9.70±0.79 ng/g, p<0.01) was found. Brain levels of DHEAS in patients with liver disease who died without HE (11.43±1.74 ng/g tissue), and in a patient who died in uremic coma (12.56 ng/g tissue) were within the control range. Increasing ALLO enhances GABAergic tonic currents concentration-dependently, but increasing DHEAS reduces these currents. High concentrations of DHEAS (50 μM) reduce GABAergic tonic currents in the presence of ALLO, whereas reduced concentrations of DHEAS (1 μM) further stimulate these currents. These findings demonstrate that decreased concentrations of DHEAS together with increased brain concentrations of ALLO increase GABAergic tonic currents synergistically; suggesting that reduced brain DHEAS could further increase GABAergic tone in human HE.     

Substantia Nigra γ-Aminobutyric Acid Receptors in Huntington''s Disease

The specific binding of [3H]gamma-aminobutyric acid (GABA) to nigral GABA receptors has been studied in postmortem brains from controls and patients with Huntington's disease (HD). A specific increase in the number of high-affinity binding sites for [3H]GABA was observed in HD patients, analogous to changes observed in rat substantia nigra [3H]GABA binding after striatal kainic acid (KA) lesion. The results provide further support for the striatal KA lesion in the rat as an animal model of HD. The implications of the results for the proposed therapeutic potential of GABA agonists in HD are discussed.     

Free Copper,Ferroxidase and SOD1 Activities,Lipid Peroxidation and NO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> Content in the CSF. A Different Marker Profile in Four Neurodegenerative Diseases

The understanding of oxidative damage in different neurodegenerative diseases could enhance therapeutic strategies. Our objective was to quantify lipoperoxidation and other oxidative products as well as the activity of antioxidant enzymes and cofactors in cerebrospinal fluid (CSF) samples. We recorded data from all new patients with a diagnosis of either one of the four most frequent neurodegenerative diseases: Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD) and lateral amyotrophic sclerosis (ALS). The sum of nitrites and nitrates as end products of nitric oxide (NO) were increased in the four degenerative diseases and fluorescent lipoperoxidation products in three (excepting ALS). A decreased Cu/Zn-dependent superoxide dismutase (SOD) activity characterized the four diseases. A significantly decreased ferroxidase activity was found in PD, HD and AD, agreeing with findings of iron deposition in these entities, while free copper was found to be increased in CSF and appeared to be a good biomarker of PD.     

Quantification of the GABA Shunt and the Importance of the GABA Shunt Versus the 2-Oxoglutarate Dehydrogenase Pathway in GABAergic Neurons

Abstract: We investigated the activity of the cerebral GABA shunt relative to the overall cerebral tricarboxylic acid (TCA) cycle and the importance of the GABA shunt versus 2-oxoglutarate dehydrogenase for the conversion of 2-oxoglutarate into succinate in GABAergic neurons. Awake mice were dosed with [1-¹³C]glucose, and brain extracts were analyzed by ¹³C NMR spectroscopy. The percent enrichments of GABA C-2 and glutamate C-4 were the same: 5.0 ± 1.6 and 5.1 ± 0.2%, respectively (mean ± SD). This, together with previous data, indicates that the flux through the GABA shunt relative to the overall cerebral TCA cycle flux equals the GABA/glutamate pool size ratio, which in the mouse is 17%. It has previously been shown that under the experimental conditions used in this study, the ¹³C labeling of aspartate from [1-¹³C]glucose specifically reflects the metabolic activity of GABAergic neurons. In the present study, the reduction in the formation of [¹³C]aspartate during inhibition of the GABA shunt by γ-vinyl-GABA indicated that not more than half the flux from 2-oxoglutarate to succinate in GABAergic neurons goes via the GABA shunt. Therefore, because fluxes through the GABA shunt and 2-oxoglutarate dehydrogenase in GABAergic neurons are approximately the same, the TCA cycle activity of GABAergic neurons could account for one-third of the overall cerebral TCA cycle activity in the mouse. Treatment with γ-vinyl-GABA, which increased GABA levels dramatically, caused changes in the ¹³C labeling of glutamate and glutamine, which indicated a reduction in the transfer of glutamate from neurons to glia, implying reduced glutamatergic neurotransmission. In the most severely affected animals these alterations were associated with convulsions.     

Brain neurotransmitter deficits in mice transgenic for the Huntington's disease mutation

Huntington's disease (HD) is associated with an expansion in the CAG repeat sequence of a gene on chromosome 4, resulting in a neurodegenerative process particularly affecting the striatum and with profound but selective changes in content of various neurotransmitters. Recently, transgenic mice expressing a fragment of the human HD gene containing a large CAG expansion have been generated; these mice exhibit a progressive neurological phenotype that includes motor disturbances, as well as neuronal deficits. To investigate their underlying neurotransmitter pathology, we have determined concentrations of GABA, glutamate, and the monoamine neurotransmitters in several brain regions in these mice and control animals at times before and after the emergence of the behavioural phenotype. In contrast to the findings in HD, striatal GABA was unaffected, although a deficit was observed in the cerebellum, consistent with a dysfunction of Purkinje cells. Losses of the monoamine transmitters were observed, some of which are not seen in HD. Thus, 5-hydroxytryptamine and, to a greater extent, 5-hydroxyindoleacetic acid levels were diminished in all brain regions studied, and noradrenaline was particularly affected in the hippocampus. Dopamine was decreased in the striatum in older animals, parallelling evidence for diminished dopaminergic activity in HD.     

Interrelationships between ornithine,glutamate, and GABA. II. Consequences of inhibition of GABA-T and ornithine aminotransferase in brain

The objective of the present study was to compare the effects of elevation of GABA concentration and those of inactivation ofl-ornithine: 2-oxoacid aminotransferase (OAT) on the in vivo metabolism ofl-ornithine (Orn) in brain. Vigabatrin (4-aminohex-5-enoic acid) and gabaculine (5-amino-1,3-cyclohexadienyl carboxylic acid), two well known inactivators of GABA-T, were used to elevate brain GABA concentrations. The latter inactivates OAT also. Transamination of Orn is, from a quantitative point of view, a significant reaction in mouse brain. GABA is a feed-back regulator of OAT. Within GABAergic neurons Orn concentration may be regulated by endogenous GABA. Extensive inactivation of OAT causes a considerable increase of Orn concentration, both in synaptosomes and in non-synaptosomal compartments. The results are compatible with a role of Orn as precursor of glutamate and/or GABA in certain neurons.     

Increased gamma-aminobutyric acid (GABA), homocarnosine and beta-alanine in cerebrospinal fluid of patients treated with gamma-vinyl GABA (4-amino-hex-5-enoic acid)

γ-Vinyl GABA, an enzyme-activated irreversible inhibitor of GABA transaminase (GABA-T), was administered orally to 15 patients with various neurological conditions at daily doses of 0.5, 1, 2 or 6 g/day for 3 days. CSF samples were obtained by lumbar puncture before treatment and within 24 hours after the last dose and the CSF concentrations of free GABA, total GABA, homocarnosine, β-alanine and γ-vinyl GABA determined by ion-exchange chromatography with fluorometric detection. γ-Vinyl GABA treatment produced dose-dependent increases in free GABA, conjugated GABA (defined as total minus free GABA), homocarnosine and β-alanine. The concentrations of CSF γ-vinyl GABA also depended on the dose administered. These results indicate that γ-vinyl GABA enters the CNS after oral administration and alters GABA metabolism by inhibition of GABA-T and suggest that such treatment may achieve therapeutic benefit in conditions where such neurochemical alterations are desirable.     

Effects of anticonvulsants on cholinergic and GABAergic properties in the neuronal cell clone NG108-15

The effects of anticonvulsant drugs on growth, cholinergic, and GABAergic properties were examined in the neuronal cell clone NG108-15. Cells were exposed for 4 days to valproic acid, phenobarbital, phenytoin, or carbamazepine in concentrations equivalent to therapeutic free levels in human serum. Experiments were also performed with varying concentrations of a recently proposed antiepileptic, gamma-vinyl GABA. Of these five anticonvulsants, cell growth (total protein and cell counts) was decreased with valproic acid and phenytoin but only valproic acid and gamma-vinyl GABA altered neurotransmitter markers. Therapeutic concentrations of valproic acid increased choline acetyltransferase activity to 142% of control but had no effect on either the activity of glutamate decarboxylase or the level of GABA. The effects of a higher (toxic) concentration of valproic acid (200 g/ml) were similar to those induced by the differentiating agent dibutyryl cyclic AMP: both decreased cell growth, enhanced the activity of choline acetyltransferase and reduced the activity of glutamate decarboxylase. Gamma-vinyl GABA had no effect on cholinergic markers but, at 1300 g/ml, increased GABA levels to 135% of control despite the reduction of glutamate decarboxylase to 68% of control. In the NG108-15 cell clone, anticonvulsants have varying effects on cell growth, differentiation, and neurotransmitter systems. Our findings do not support the proposal that the mechanism of action for valproic acid, phenobarbital, phenytoin, and carbamazepine is via alteration of GABA levels.     

Concentration Gradients of Free and Total γ-Aminobutyric Acid and Homocarnosine in Human CSF: Comparison of Suboccipital and Lumbar Sampling

Abstract: Concentrations of free and total γ-aminobutyric acid (GABA) and homocarnosine were determined in sequential aliquots of the first 30 ml of CSF obtained by lumbar puncture in five patients. Rostrocaudal gradients were calculated and compared to gradients estimated by determining concentrations of these substances in CSF obtained by simultaneous suboccipital and lumbar punctures in four more patients. In the lumbar fractions study, rostrocaudal mean gradients of 0.36, 36, and 21 pmol/ml for free GABA, total GABA, and homocarnosine, respectively, were calculated. In the suboccipital/lumbar study, gradients of 0.33, 30, and 24 pmol/ml for free GABA, total GABA, and homocarnosine, respectively, were estimated. These results indicate that valid comparison of CSF concentrations of these substances is restricted to similar fractions and suggest that in CSF the substances originate largely from brain rather than from peripheral sources.     

Regional Variation in γ-Aminobutyric Acid Turnover: Effect of Castration on γ-Aminobutyric Acid Turnover in Microdissected Brain Regions of the Male Rat

Abstract: This study compared the turnover of GABA neurons in different brain areas of the male rat and examined the effect of castration on GABA turnover in regions of the brain associated with the control of gonadotropin secretion. To estimate GABA turnover, GABA was quantified by HPLC in microdissected brain regions 0,30,60,90, and 120 min after inhibition of GABA degradation by aminooxyacetic acid (100 mg/kg, i.p.). GABA accumulation was linear in all areas for 90 min ( p < 0.01), and GABA turnover was estimated as the slope of the line formed by increased GABA concentration versus time, determined by linear regression. There was considerable regional variation both in the initial steady-state concentrations of GABA and in the rates of GABA turnover. Of 10 discrete brain structures, GABA turnover was highest in the medial preoptic nucleus and lowest in the caudate nucleus. Turnover times in the terminal fields of known GABAergic projection neurons ranged sevenfold, from 2.6 h in the substantia nigra to 0.4 h in the lateral vestibular nucleus. The effect of castration on GABA turnover in 13 microdissected brain regions was investigated by measuring regional GABA concentrations before and 30 min after injection of aminooxyacetic acid in intact rats or 2 or 6 days postcastration. Following castration, steady-state GABA concentrations were increased, and GABA turnover decreased in the diagonal band of Broca, the medial preoptic area, and the median eminence. GABA turnover increased in the medial septal nucleus and was unaffected in the cortex, striatum, and hindbrain. These results are consistent with the hypothesis that testosterone negative-feedback control of luteinizing hormone-releasing hormone involves steroid-sensitive GABAergic neurons in the rostral and medial basal hypothalamus.     

Implications for treatment: GABAA receptors in aging, Down syndrome and Alzheimer's disease

In addition to progressive dementia, Alzheimer's disease (AD) is characterized by increased incidence of seizure activity. Although originally discounted as a secondary process occurring as a result of neurodegeneration, more recent data suggest that alterations in excitatory-inhibitory (E/I) balance occur in AD and may be a primary mechanism contributing AD cognitive decline. In this study, we discuss relevant research and reports on the GABA(A) receptor in developmental disorders, such as Down syndrome, in healthy aging, and highlight documented aberrations in the GABAergic system in AD. Stressing the importance of understanding the subunit composition of individual GABA(A) receptors, investigations demonstrate alterations of particular GABA(A) receptor subunits in AD, but overall sparing of the GABAergic system. In this study, we review experimental data on the GABAergic system in the pathobiology of AD and discuss relevant therapeutic implications. When developing AD therapeutics that modulate GABA it is important to consider how E/I balance impacts AD pathogenesis and the relationship between seizure activity and cognitive decline.     

Pharmacological and Biochemical Aspects of GABAergic Neurotransmission: Pathological and Neuropsychobiological Relationships

1. The GABAergic neurotransmission has been implicated in the modulation of many neural networks in forebrain, midbrain and hindbrain, as well as, in several neurological disorders.2. The complete comprehension of GABA system neurochemical properties and the search for approaches in identifying new targets for the treatment of neural diseases related to GABAergic pathway are of the extreme relevance.3. The present review will be focused on the pharmacology and biochemistry of the GABA metabolism, GABA receptors and transporters. In addition, the pathological and psychobiological implications related to GABAergic neurotransmission will be considered.     

Comparative effects of the GABA uptake inhibitors,tiagabine and NNC-711, on extracellular GABA levels in the rat ventrolateral thalamus

The primary mechanism by which the action of synaptically released GABA is thought to be terminated is by re-uptake into neurones and glial cells, and the pharmacological inhibition of this uptake may be beneficial in conditions where decreased GABAergic transmission has been implicated, such as epilepsy. We have compared the effects of two of these uptake inhibitors, tiagabine and NNC-711, on extracellular GABA levels in the thalamus of the rat, after both systemic and local administration. Both compounds produced dose-dependent increases in GABA concentration irrespective of the route of administration, but the concentrations required to produce increased extracellular GABA levels were considerably higher than those known to be effective for anticonvulsant purposes. These data suggest that, initially at least, alternative GABA transporters, not susceptible to inhibition by the compounds used, may still be able to remove synaptically released GABA from the extracellular space. Special issue dedicated to Dr. Kinya Kuriyama.