γ-Hydroxybutyric Acid in Subcellular Fractions of Rat Brain

The presence of gamma-hydroxybutyric acid (GHB) in synaptosome-enriched fractions of rat brain was ascertained using a GLC technique. The stability of GHB in synaptosomes was evaluated by addition of various gamma-aminobutyric acid (GABA) transaminase (GABA-T) inhibitors, GHB, or ethosuximide to the homogenizing medium. Furthermore, changes in whole brain GHB levels were compared with those in the synaptosomal fraction in animals treated with GABA-T inhibitors, GABA, or ethosuximide. GHB was present in synaptosome-enriched fractions in concentrations ranging from 40 to 70 pmol/mg of protein. There was no evidence for redistribution, leakage, or metabolism of GHB during the preparation of synaptosomes. The elevations of whole brain GHB level associated with GABA-T or ethosuximide treatment were reflected by a parallel increase in synaptosomal GHB content. These data add to the growing evidence that GHB may have neurotransmitter or neuromodulator function.     

Changes in the Amino Acid Content of Nerve Endings (Synaptosomes) Induced by Drugs that Alter the Metabolism of Glutamate and γ-Aminobutyric Acid

The study was centered on the changes in the amino acid content of nerve endings (synaptosomes) induced by drugs that alter the metabolism of glutamate or gamma-aminobutyric acid (GABA), and that possess convulsant or anticonvulsant properties. The onset of seizures induced by various convulsant agents was associated with a decreased content of GABA and an increased content of glutamate in synaptosomes. The concurrent administration of pyridoxine prevented both the biochemical changes and the convulsions. The administration of gabaculine to mice resulted in large increases in the GABA content of synaptosomes that were counteracted by decreases in glutamate, glutamine, and aspartate levels such that the total content of the four amino acids remained unchanged. The administration of aminooxyacetic acid (0.91 mmol/kg) resulted initially in seizure activity, but subsequently in an anticonvulsant action. No simple relationship existed between the excitable state of the brain induced by aminooxyacetic acid and the changes in the synaptosomal levels of any of the amino acid transmitters. A hypothesis was, however, formulated that explained the convulsant-cum-anticonvulsant action of aminooxyacetic acid on the basis of compartmentation of GABA within the nerve endings.     

Interactions of Di-n-Propylacetate, Gabaculine, and Aminooxyacetic Acid: Anticonvulsant Activity and the γ-Aminobutyrate System

Abstract: Di-n-propylacetate (DPA), aminooxyacetic acid (AOAA), and gabaculine were administered alone or in combination to Swiss mice. Six hours after administration of the drugs the anticonvulsant action (against isonicotinic acid hydrazide-induced seizures) of AOAA and DPA combined was less than that of AOAA alone. The cause of this phenomenon appeared to be an interaction between DPA and AOAA with respect to inhibition of GABA-T activity, resulting in a long-term diminished inhibition by AOAA, which in turn led to a lessening of the AOAA-induced elevation in the GABA content of nerve endings (synaptosomes). An excellent correlation was observed between the delay in onset of seizures and the elevation of synaptosomal GABA content.     

Improved Method for Isolating Synaptosomes from 11 Regions of One Rat Brain: Electron Microscopic and Biochemical Characterization and Use in the Study of Drug Effects on Nerve Terminal γ-Aminobutyric Acid in Vivo

A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3-phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll-sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional gamma-aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3-mercaptopropionic acid (3-MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GABA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium-free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA-synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3-MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3-MP-induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3-MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.     

Stimulation of Synaptosomal γ-Aminobutyric Acid Synthesis by Glutamate and Glutamine

gamma-Aminobutyric acid (GABA) synthesis was studied in rat brain synaptosomes by measuring the increase of GABA level in the presence of the GABA-transaminase inhibitor gabaculine. The basal rate of synaptosomal GABA synthesis in glucose-containing medium (25.9 nmol/h/mg of protein) was only 3% of the maximal activity of glutamate decarboxylase (GAD; 804 +/- 83 nmol/h/mg of protein), a result indicating that synaptosomal GAD operates at only a small fraction of its catalytic capacity. Synaptosomal GABA synthesis was stimulated more than threefold by adding 500 microM glutamine. Glutamate also stimulated GABA synthesis, but the effect was smaller (1.5-fold). These results indicate that synaptosomal GAD is not saturated by endogenous levels of its substrate, glutamate, and account for part of the unused catalytic capacity. The greater stimulation of GABA synthesis by glutamine indicates that the GAD-containing compartment is more accessible to extrasynaptosomal glutamine than glutamate. The strong stimulation by glutamine also shows that the rates of uptake of glutamine and its conversion to glutamate can be sufficiently rapid to support GABA synthesis in nerve terminals. Synaptosomes carried out a slow net synthesis of aspartate in glucose-containing medium (7.7 nmol/h/mg of protein). Aspartate synthesis was strongly stimulated by glutamate and glutamine, but in this case the stimulation by glutamate was greater. Thus, the larger part of synaptosomal aspartate synthesis occurs in a different compartment than does GABA synthesis.     

EFFECT OF γ-AMINOBUTYRIC ACID ON BRAIN SEROTONIN AND CATECHOLAMINES

—Intraperitoneal injections of GABA (5 mg/kg) to rats lowered the level of norepinephrine in brain, heart and spleen but not suprarenals and raised that of serotonin in brain. Changes of these monoamines were most pronounced in the hypothalamic region after 20min. A reduction of hypothalamic norepinephrine was also observed 15min following the intracarotid administration of 0·5 mg/kg of GABA. In these experiments there was a concomitant increase in the level of free GABA in the anterior portion of the ventral hypothalamus. Brain dopamine level and 5-hydroxytryptophan decarboxylase, dihydroxyphenylalanine decarboxylase and monoamine oxidase activities were not affected. The 20 per cent increase of endogenous GABA observed in the midbrain 30 min following the administration of amino-oxyacetic acid was accompanied by a sharp fall in norepinephrine level (39 per cent) and an increase in serotonin (20 per cent). In in vitro studies 10–300 μg/ml of GABA were shown to release norepinephrine from cortical and hypothalamic slices, and to inhibit serotonin release without affecting 5-hydroxytryptophan uptake and to have no effect on the release of dopamine from slices of the region of the corpus striatum nor on the activity of the enzymes mentioned. Subcellular studies showed that the particulate:supernatant ratio for norepinephrine was reduced from a control value of 2·04 to 1·75 and that of serotonin was raised from 2·8 to 3·5. Following pretreatment with iproniazid, GABA reduced the raised level of brain norepinephrine to a greater extent than reserpine but not as intensively as amphetamine. The results obtained suggest that these monoamines may be involved in the mechanisms underlying the action of GABA in brain and that the effect of GABA on brain monoamines may be of certain significance in synaptic events.     

HYDRAZIDE-INDUCED SEIZURES AND CEREBRAL OF LEVELS γ-AMINOBUTYRIC ACID: A RE-EVALUATION

Data have been presented indicating that in chicks the time lag in the onset of seizures after the administration of hydrazides was not caused by a slow penetration of the convulsant agents into the brain. A correlation was evident between the rate of decrease in concentration of cerebral GABA and the susceptibility of chicks to hydrazide-induced seizures. Earlier studies precluding a role for GABA in hydrazide-induced convulsions were re-examined.     

Human Brain γ-Aminobutyric Acid Levels and Seizure Control Following Initiation of Vigabatrin Therapy

Abstract: Vigabatrin is a novel antiepileptic drug designed to control seizures by raising brain γ-aminobutyric acid (GABA) concentrations. Seizure control is not improved significantly when the daily dose is increased beyond 50 mg/kg. Serial, in vivo measurements of GABA levels in human occipital lobe were made using ¹H NMR spectroscopy before and after the start of vigabatrin treatment. We used a 2.1-T magnetic resonance imagerspectrometer and an 8-cm surface coil to examine serially a 14-cm³ volume in the occipital lobe of 26 patients with complex partial seizures. Brain GABA content increased following the start of vigabatrin treatment up to a daily dose of 60 mg/kg. Additional increases in dose failed to increase brain GABA content further. GABA synthesis may decrease with sustained elevations of human brain GABA levels. Starting vigabatrin treatment reduced seizure frequency by >50%, from six to seven per month to three. Improved seizure control was not associated with further increases of vigabatrin dose. Increased brain GABA concentration was associated with improved seizure control. Starting vigabatrin treatment improved seizure control twofold when GABA levels increased above 1.8 mmol/kg. Further increases in brain GABA content above 2.5 mmol/kg provided less protection. Measuring occipital lobe GABA concentrations may predict improved seizure control when using antiepileptic drugs designed to increase brain GABA levels.     

Cerebral Glutamic Acid Decarboxylase Activity and γ-Aminobutyric Acid Concentrations in Mice Susceptible or Resistant to Audiogenic Seizures

Abstract: DBA/2 mice between 21 and 28 days of age are highly susceptible to sound-induced seizures. Drug studies suggest a possible deficit of γ-Aminobutyric acid (GABA)-mediated neurotransmission may be involved. We have measured the whole brain GABA concentration and glutamic acid decar-boxylase activity in DBA/2 mice at various ages before, during, and after the period of maximal susceptibility to audiogenic seizures. Corresponding determinations were carried out on age-matched TO mice, a strain much less susceptible to audiogenic seizures than DBA/2 mice at all ages. No significant differences in GABA concentration or glutamic acid decarboxylase activity were found between strains at any age. The susceptibility of DBA/2 mice to audiogenic seizures does not result from a gross inability to synthesise or store GABA.     

Uptake of γ-Aminobutyric Acid by Brain Tissue Preparations: A Reevaluation

The kinetic constants Km and Vmax for the uptake of gamma-aminobutyric acid (GABA) by various preparations from rat cerebral cortex were determined by means of Eadie-Hofstee plots and computer analysis. The Km values were much greater in 0.1-mm slices than in synaptosomal preparations, and the Km value increased further with the thickness of the slices. The apparent high Km values in slices were probably due to depletion of the GABA concentration in the extracellular fluid as the exogenous GABA ran the gauntlet of competing uptake sites on its way to sites deep within the slice, thereby bringing about a requirement for higher GABA concentrations in the incubation medium in order to maintain the internal GABA levels at the "Km level." Evidence was obtained for three GABA uptake systems with Km values (in synaptosomes) of 1.1 microM, 43 microM, and 3.9 mM, respectively. In contrast, only two uptake systems for D-aspartate were detected, with Km values of 1.8 microM and 1.8 mM, respectively. The implications of the findings in the study with respect to previous data in the literature are discussed.     

The Level of GAD67 Protein Is Highly Sensitive to Small Increases in Intraneuronal γ-Aminobutyric Acid Levels

Increases (>2.5-fold) in GABA levels in rat brain lead to a large decrease in the level of the 67-kDa form of glutamate decarboxylase (GAD₆₇) through a mechanism involving either a change in GAD₆₇ protein stability or a change in GAD₆₇ mRNA translation. In the present study, brain levels of GABA were manipulated by treating rats with various doses of γ-vinyl-γ-aminobutyric acid (GVG), and the dependence of total GAD activity and levels of GAD₆₇ and GAD₆₅ protein on the levels of GABA was analyzed. Initial studies showed that both GABA and GAD₆₇protein levels reached new steady-state levels after two to four daily injections; GABA increased 1.5- (30 mg of GVG/kg) and fourfold (150 mg of GVG/kg), and GAD₆₇ protein content decreased by 30 and 70%. To assess the sensitivity of GAD₆₇ to GABA, rats were injected with eight different doses of GVG (15-150 mg/kg) for 5 days. With increasing doses of GVG, we observed a gradual increase in both whole-tissue and synaptosomal GABA levels and a gradual decrease in GAD₆₇ protein and GAD activity. The levels of GAD₆₇ remained constant at all GVG doses. GAD₆₇ was remarkably sensitive to GABA. The synaptosomal GAD₆₇ level decreased ∼12% and the whole-neuron GAD₆₇ level decreased ∼3% for each 1 % increase in nerve terminal GABA content when it was close to its physiological level. Our results clearly demonstrate that GAD₆₇ is tightly controlled by intraneuronal GABA, and we suggest that this regulatory mechanism has important implications for the physiological regulation of GABAergic function in the mammalian brain.     

THE COMPARATIVE EFFECTS OF VARIOUS HYDRA-ZIDES ON γ-AMINOBUTYRIC ACID AND ITS METABOLISM

Abstract— The administration of different hydrazides to chicks (20-23 days post-hatching) in amounts giving similar latent periods before the onset of seizures produced (i) similar rates of decrease in content of cerebral GABA, (ii) considerable but dissimilar inhibitions of cerebral GAD activity, (iii) slight inhibitions of cerebral GABA-T activity. The results support the view that low GABA levels were involved in. the etiology of the seizures but seemed to rule out the possibility that a reduced turnover of GABA was responsible for the occurrence of the convulsions.     

Reversible Alterations of Cerebral γ-Aminobutyric Acid in Pyrithiamine-Treated Rats: Implications for the Pathogenesis of Wernicke''s Encephalopathy

Treatment of rats with the central thiamine antagonist, pyrithiamine, results in severe neurological symptoms such as loss of righting reflex. Measurement of gamma-aminobutyric acid (GABA) content of brain tissue from symptomatic pyrithiamine-treated (PT) rats revealed significant reductions in thalamus, cerebellum, and pons. GABA content of cerebral cortex, however, was unaltered. Activities of the thiamine-dependent enzyme alpha-ketoglutarate dehydrogenase (alpha KGDH) were reduced in parallel with the GABA changes. On the other hand, activities of the GABA-synthetic enzyme glutamic acid decarboxylase (GAD) remained within normal limits, with the exception of a small but significant decrease in thalamus of symptomatic PT rats. Affinities and densities of high-affinity [3H]muscimol binding sites on crude cerebral membrane preparations from symptomatic PT rats were unchanged. Thiamine administration to symptomatic animals resulted in correction of abnormal righting reflexes and in normalization of decreased GABA levels and reduced alpha KGDH activities in cerebellum and pons. Thalamic GABA levels and alpha KGDH activities, on the other hand, remained significantly lower than normal. These results suggest that the reversible symptoms of pyrithiamine treatment may result from imparied GABA synthesis in cerebellum and pons of these animals. Similar mechanisms may play a role in the pathogenesis of the reversible symptoms of Wernicke's encephalopathy in man.     

Tissue Distribution, Metabolism, Anticonvulsant Efficacy, and Effect on Brain Amino Acid Levels of the Glia-Selective γ-Aminobutyric Acid Transport Inhibitor 4,5,6,7-Tetrahydroisoxazolo[4,5-c]pyridin-3-ol in Mice and Chicks

Using tritium-labelled 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) its tissue distribution and metabolism were investigated in adult mice and 4-day-old chicks after systemic administration of the drug. It was found not to be significantly metabolized in the brain since metabolites of THPO corresponding to only approximately 8% of the parent compound could be detected 30 min after administration of the drug intramuscularly in mice. In the liver, however, THPO was found to be metabolized to a considerable extent. In chicks THPO metabolites were found in the brain but they accounted for less than 35% of the radioactivity. The brain concentration of THPO in mice and chicks corresponded to respectively 10 and 50% of the dose injected intramuscularly and the tissue level was essentially constant for at least 3 h after injection. Following systemic administration of THPO to mice and chicks the contents of aspartate, glutamate, glutamine, and gamma-aminobutyric acid (GABA) in whole brain and in synaptosomes was determined. It was found that only GABA contents were affected being increased in synaptosomes from mice and decreased in whole brain in chicks. Doses of THPO, which in chicks but not in mice led to brain levels that were sufficient to inhibit glial GABA uptake, were found to protect chicks but not mice against isonicotinic acid hydrazide-induced seizures. The findings are compatible with the notion that THPO exerts its anticonvulsant activity by inhibition of astrocytic GABA uptake.     

Differences in Anionic Dependence of the Synaptic Efflux of d-Aspartic Acid and γ-Aminobutyric Acid

The synaptosomal effluxes of D-aspartate and gamma-aminobutyric acid (GABA) induced by a substitution of the Cl- ions with propionate in the incubation medium were measured in preparations of hippocampal tissue homogenates. The efflux of aspartate was significantly higher than spontaneous efflux at 125 mM Cl- (normal = 144 mM) and was increased with decreasing Cl- concentration. GABA efflux was much less sensitive to a reduction in Cl- concentration than D-aspartate. The efflux was Ca2+-dependent in both cases.     

Enhancement of γ-Aminobutyric Acid Binding by the Anxiolytic β-Carbolines ZK 93423 and ZK 91296

The effects of two anxiolytic beta-carboline derivatives, ZK 93423 and ZK 91296, on the binding of gamma-[3H]aminobutyric acid ([3H]GABA) to brain membrane preparations from rat cerebral cortex were examined. ZK 93423 concentration-dependently enhanced the specific binding of [3H]GABA, with a maximal increase of 45% above control at a 50 microM concentration. A less pronounced increase was induced by diazepam and by the partial agonist ZK 91296. Scatchard plot analysis revealed that the effect of ZK 93423 was due to an increase in the total number of high- and low-affinity GABA binding sites. The action of ZK 93423 was mediated by benzodiazepine recognition sites since it was blocked by the benzodiazepine antagonists Ro 15-1788 and ZK 93426 at concentrations that failed to modify [3H]GABA binding on their own. Moreover the stimulatory effect of ZK 93423 on [3H]GABA binding was also blocked by the beta-carboline inverse agonist ethyl beta-carboline-3-carboxylate. These results are consistent with the view that ZK 93423 and ZK 91296, similarly to benzodiazepines, exert their pharmacological effects by enhancing the GABAergic transmission at the level of the GABA/benzodiazepine receptor complex.     

REGIONAL γ-AMINOBUTYRIC ACID LEVELS IN RAT BRAIN DETERMINED AFTER MICROWAVE FIXATION

—GABA levels in rat whole brain were compared following three methods of sacrifice: rapid microwave fixation, decapitation into liquid nitrogen, and decapitation at 20°C. Levels were shown to be identical in animals sacrificed by microwave fixation and decapitation into liquid nitrogen. In contrast, rats decapitated at 20°C had 18 per cent higher GABA levels when determined immediately post-mortem and 48 per cent higher levels after 30 min at 20°C. Microwave treatment prevented these post-mortem increases. The increase in GABA after decapitation at 20°C was even greater in hypothalamus than in whole brain. A comparison of 3 GABA extraction methods following microwave fixation demonstrated that sodium acetate was 88 per cent as effective as 80 per cent ethanol and more effective than 0·5 n -perchloric acid in extracting GABA. Fifteen brain regions were dissected from microwave-treated brains and the GABA levels determined.     

Regulation of Transmitter γ-Aminobutyric Acid (GABA) Synthesis and Metabolism Illustrated by the Effect of γ-Vinyl GABA and Hypoglycemia

The effect of different treatments on amino acid levels in neostriatum was studied to throw some light on the synthesis and metabolism of gamma-aminobutyric acid (GABA). Irreversible inhibition of GABA transaminase by microinjection of gamma-vinyl GABA (GVG) led to a decrease in aspartate, glutamate, and glutamine levels and an increase in the GABA level, such that the nitrogen pool remained constant. The results indicate that a large part of brain glutamine is derived from GABA. Hypoglycemia led to an increase in the aspartate level and a decrease in glutamate, glutamine, and GABA levels. The total amino acid pool was decreased compared with amino acid levels in normoglycemic rats. GVG treatment of hypoglycemic rats led to a decrease in the aspartate level and a further reduction in glutamate and glutamine levels. In this case, GABA accumulation continued, although the glutamine pool was almost depleted. The GABA level increased postmortem, but there were no detectable changes in levels of the other amino acids. Pretreatment of the rats with hypoglycemia reduced both glutamate and glutamine levels with a subsequent decreased postmortem GABA accumulation. The half-maximal GABA synthesis rate was obtained when the glutamate level was reduced by 50% and the glutamine level was reduced by 80%.     

Catecholamine Concentration in the Developing Rat Brain after γ-Hydroxybutyric Acid

The effect of the GABA receptor agonist γ-hydroxybutyric acid (GHBA) on brain catecholamine concentration was investigated in 1 to 28 day old rats. The infant rats were given GHBA in various doses (375–1500 mg/kg) and the effects on whole brain or regional brain concentration of dopamine (DA) and noradrenaline (NA) were measured. Brain DA concentration increased in a dose-dependent way already from two days of postnatal age. In the regional brain study of the 14- and 28-day-old animals the increase in DA concentration was found to be almost exclusively located in the striatal region. Generally, no changes in NA concentration were found in the whole brain or various brain regions at any of the ages after GHBA. It is concluded that the inhibitory striatal-nigral neurons, utilizing GABA as a transmitter, are functionally developed during early postnatal age.     

Administration of Vigabatrin (γ-Vinyl-γ-Aminobutyric Acid) Affects the Levels of Both Inhibitory and Excitatory Amino Acids in Rat Cerebrospinal Fluid

The effect of vigabatrin (gamma-vinyl-gamma-aminobutyric acid), a new anticonvulsant drug, on the transmitter amino acids in rat cisternal CSF was studied. CSF was collected through a permanently implanted polyethylene cannula from freely moving rats at 5, 24, 48, and 96 h after administration of 1,000 mg/kg of vigabatrin. The free gamma-aminobutyric acid (GABA) level was elevated maximally (13.5-fold; p less than 0.01) at 24 h after injection. The homocarnosine (GABA-histidine) level also was increased (123%; p less than 0.01) at 24 h after injection, and its concentration remained at the same level for the next 3 days. Glycine and taurine concentrations had increased [31% (p less than 0.05) and 63% (p less than 0.01), respectively] at 5 h after injection. It is interesting that the levels of glutamate and aspartate increased [330% (p less than 0.05) and 421% (p less than 0.01), respectively] at 96 h after injection, the time when the free GABA level had returned to the baseline concentration and the vigabatrin level was 3% of the maximal concentration. The present study indicates that a single dose of vigabatrin in rats elevates levels of both the inhibitory and excitatory amino acids in CSF. However, the temporal profile of observed changes in relation to vigabatrin injection shows that neither the long-lasting elevation of GABA content nor the increase in glutamate and aspartate levels correlates with the level of vigabatrin in CSF. These findings suggest that the excitatory mechanisms are also augmented following acute administration of vigabatrin, especially when the content of GABA had decreased to the baseline level and the level of vigabatrin was low.