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.     

Distribution of Activity Converting 4-Aminobutyraldehyde to γ-Aminobutyric Acid in Subcellular Fractions of Mouse Brain

The subcellular distribution of activity of 4-aminobutyraldehyde dehydrogenase (ABAL-DH) was studied in mouse brain. ABAL-DH was localized mainly in the crude mitochondrial fraction; most of the activity in this fraction was found in the subfraction containing synaptosomes, and the remainder was in the mitochondrial fraction. After osmotic disruption of synaptosomes, most of the activity was located in the synaptic cytosol, and the remainder was in the synaptic mitochondria. Sucrose density subfractionation of synaptosomes revealed that gamma-aminobutyric acid, glutamic acid decarboxylase, and ABAL-DH localized in a denser region of gradient fraction than the region containing acetylcholinesterase and choline acetyltransferase.     

Influence of Sodium-Independent γ-Aminobutyric Acid Binding on the Assay of Sodium-Dependent γ-Aminobutyric Acid Binding in a Membrane Preparation of Rat Brain

A large amount of [³H]GABA was bound to crude synaptic membrane fractions of rat. by sodium-independent process in a medium that contained 100 μM [³H]GABA used for assaying GABA uptake site. This [³H]-GABA binding was different from receptor binding of GABA. It was confirmed that this sodium-independent [²H]GABA binding scarcely occurred in the presence of a physiological concentration of sodium chloride, and that sodium-independent GABA binding had a negligible influence on sodium-dependent GABA binding.     

Uptake of γ-Aminobutyric Acid by a Synaptic Vesicle Fraction Isolated from Rat Brain

Gamma-Aminobutyric acid (GABA) was taken up by a MgATP-dependent mechanism into synaptic vesicles isolated by hypoosmotic shock and density gradient centrifugation. The properties of the vesicular uptake differed clearly from those of synaptosomal and glial uptake, both with respect to Na+, Mg2+, and ATP dependence and with respect to response to general GABA uptake inhibitors such as nipecotic acid, diaminobutyric acid, and beta-alanine. The uptake showed a Km of 5.6 mM and a net uptake rate of 1,500 pmol/min/mg of protein. It is suggested that the vesicular uptake of GABA is driven by an electrochemical proton gradient generated by a Mg2+-ATPase.     

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.     

γ-Glutamylaminotransferase and Transglutaminase in Subcellular Fractions of Rat Cortex and in Cultured Astrocytes

The subcellular distribution of gamma-glutamylamino-transferase (EC 2.3.2.2) and transglutaminase (EC 2.3.2.1) has been investigated in rat brain tissue fractionated by a centrifugation and sedimentation technique. Neither of these enzymes was enriched in the synaptosomal fraction. Comparing the in vitro grown astrocytes with synaptosomes, we find that both of these enzymes may possibly be more important in the glial element of the synaptic region. gamma-Glutamylaminotransferase is most abundant in capillaries, confirming previous reports.     

In Vivo Changes in γ-Aminobutyric Acid Output from the Cerebral Cortex Induced by Inhibitors of γ-Aminobutyric Acid Uptake and Metabolism

Abstract: The effects of inhibitors of γ-aminobutyric acid (GABA) metabolism or uptake on GABA output from the cerebral cortex was studied by means of a collecting cup placed on the exposed cortex of rats anaesthetized with urethane. GABA was identified and quantified by a mass-fragmentographic method. Ethanolamine-O-sulphate (10⁻² M ) applied directly on the cerebral cortex caused a long-lasting twofold increase in GABA output, whereas dl -2, 4-diaminobutyric acid (5 × 10⁻³ M ) caused a sevenfold increase and β -alanine was inactive. The results indicate that glial uptake has little effect on GABA inactivation in the cerebral cortex. The inhibition of neuronal uptake seems a more effective tool to increase GABA concentration in the synaptic cleft, and consequently also in GABA output, than the inhibition of GABA metabolism.     

γ-Aminobutyric Acid Receptor Ionophore Complexes: Differential Effects of Deltamethrin, Dichlorodiphenyltrichloroethane, and Some Novel Insecticides in a Rat Brain Membrane Preparation

The binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS), a gamma-aminobutyric acid (GABA)-activated chloride ionophore ligand; [3H]diazepam, a benzodiazepine agonist; and [3H]muscimol, a GABA receptor probe, were used to assess the effects at 100 microM of deltamethrin, dichlorodiphenyltrichloroethane (DDT), and three experimental insecticides--a DDT-pyrethroid hybrid, GH414 (cycloprothrin), and two DDT-analogues, GH266 and GH149 (EDO), on GABA receptor ionophore complexes in a rat brain membrane preparation. GH266 and GH149 were found to inhibit a greater percentage of [35S]TBPS binding than the same concentration of deltamethrin or DDT, although GH414 had little effect. GH266 and GH149 enhanced [3H]diazepam binding by nearly 200%, in contrast to the inhibitory effects of deltamethrin, DDT, and GH414. GH266 and GH149 also caused a dramatic enhancement of [3H]muscimol binding, 367 and 236% of control, respectively, whereas DDT and deltamethrin caused only a moderate enhancement. The effects of the insecticides on binding affinity and density were examined for each of the ligands. The results show a differential interaction of the insecticides on the various ligand binding sites.     

Regional and Subcellular Localization in Rat Brain of the Enzymes That Can Synthesize γ-Hydroxybutyric Acid

Abstract: Rat brain contains two major NADPH-linked aldehyde reductases that can reduce succinate semialdehyde to 4-hydroxybutyrate. One of these enzymes appears to be fairly specific for succinate semialdehyde and is not significantly inhibited by classic aldehyde reductase inhibitors such as barbiturates. The other enzyme can reduce several aromatic aldehydes and is strongly inhibited by barbiturates and branched-chain fatty acids. Using one such inhibitor, it was possible to distinguish between and measure the two enzyme activities separately in various rat brain regions and in subcellular fractions. Both enzymes are mainly cytoplasmic but there is some activity in the synaptosomal fraction. The activity of the specific succinic semialdehyde reductase is highest in the cerebellum, where it represents 21% of the total activity, and lowest in the cortex, where it represents about 11% of the total activity.     

Presence of γ-Aminobutyric Acid in Rat Ovary

Abstract: As γ-aminobutyric acid (GABA) was first discovered as the free acid in the mammalian central nervous system, it has been assumed that GABA is generally to be found in significant amounts only in the brain, in spite of reports of its presence in a number of non-neuronal tissues. In this study, GABA was detected amongst the free amino acids in most rat tissues that were examined. The highest concentration outside the brain was in the ovary (0.59 μmol/g fresh tissue). It is concluded that the synthesis of the GABA is intragonadal and probably of metabolic importance.     

Early Undernutrition and [3H]γ-Aminobutyric Acid Binding in Rat Brain

The effect of early undernutrition and dietary rehabilitation on [3H]gamma-aminobutyric acid ([3H]GABA) binding in rat brain cerebral cortex and hippocampus was examined. Undernourished animals were obtained by exposing their mothers to a protein-deficient diet during both gestation and lactation. Saturation analysis of [3H]GABA binding in the cerebral cortex and hippocampus revealed high- and low-affinity components in the undernourished group, whereas control animals possessed only a low-affinity site. The concentration of low-affinity binding sites was greater in the undernourished animals. Rehabilitation of undernourished animals completely abolished the binding site differences. Treatment of brain membranes with Triton X-100 yielded two binding components in both the undernourished and control animals, although the concentration of lower affinity sites was still greater in the undernourished group. Neither the efficacy nor the potency of GABA to activate benzodiazepine binding in cerebral cortex was modified by undernutrition. These data suggest that early undernourishment modifies the characteristics of [3H]GABA binding, perhaps by reducing the brain content of endogenous inhibitors of the higher affinity binding site. The lack of effect on GABA-activated benzodiazepine binding suggests the possibility that neither the high- nor the low-affinity GABA binding sites are coupled to this receptor component.     

Effect of Early Iron Deficiency in Rat on the γ-Aminobutyric Acid Shunt in Brain

Early iron deficiency in rat does not affect the weight or the protein, DNA, and RNA content but results in a slight reduction in gamma-aminobutyric acid (GABA) (13%, p less than 0.01) and glutamic acid (20%, p less than 0.001) content of the brain. The activities of the two GABA shunt enzymes, glutamate dehydrogenase and GABA-transaminase, and of the NAD+-linked isocitrate dehydrogenase (ICDH) were inhibited whereas the glutamic acid decarboxylase, mitochondrial NADP+-linked ICDH, and succinic dehydrogenase activities remained unaltered in brain. On rehabilitation with the iron-supplemented diet for 1 week, these decreased enzyme activities in brain attained the corresponding control values. However, the hepatic nonheme iron content increased to about 80% of the control, after rehabilitation for 2 weeks. A prolonged iron deficiency resulting in decreased levels of glutamate and GABA may lead to endocrinological, neurological, and behavioral alterations.     

Regional Selectivity of a γ-Aminobutyric Acid-Induced [3H]Acetylcholine Release Sensitive to Inhibitors of γ-Aminobutyric Acid Uptake

The effects of gamma-aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with [3H]choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01-0.3 mM) the basal release of newly synthesized [3H]ACh was increased in a concentration-dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of [3H]ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of [3H]ACh release when tested at 0.3 mM whereas, at this concentration, (-)-baclofen was totally inactive. The GABA-induced release of [3H]ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of [3H]ACh following penetration into cholinergic nerve terminals through a GABA transport system.     

Deuterium Isotope Effect in γ-Aminobutyric Acid Transamination: Determination of Rate-Limiting Step

The rate of transamination of gamma-aminobutryic acid (GABA) catalyzed by hog brain gamma-aminobutyrate aminotransferase was substantially reduced when the hydrogen at the gamma-carbon position was replaced by deuterium. The deuterium isotope effect of this reaction has been substantiated by fluorometric, radiometric, and mass spectrometric procedures and assessed kinetically. The ratios of Vmax of the nonlabeled substrate/Vmax of the deuterated substrate obtained under different conditions ranged from 6 to 7. This indicates that the cleavage of the hydrogen from the gamma-carbon is the rate-determining step in GABA transamination. Similar isotope effects have also been shown to occur in the peripheral system in vivo.     

Formation of γ-Amino-β-Hydroxybutyric Acid from 2-Hydroxyputrescine in Rat Brain

gamma-Amino-beta-[3H]hydroxybutyric acid ([3H]-GABOB) was formed in rat brain from 2-[3H]-hydroxyputrescine that had been chemically synthesized from 2-oxoputrescine and [3H]sodium borohydride. After the injection of 2-[3H]hydroxyputrescine into the lateral ventricle of a rat brain, the rat was killed and then the brain was removed. [3H]GABOB in the brain was identified by a combination of ion-exchange chromatography, high-voltage paper electrophoresis, and recrystallization of the radioactive compound with authentic GABOB.     

Mass Fragmentographic Determination of γ-Aminobutyric Acid and Glutamic Acid in Discrete Amygdaloid Nuclei of Rat Brain

A mass fragmentographic method for the simultaneous quantification of gamma-aminobutyric acid (GABA) and glutamic acid is described. In a convenient one-step reaction, the two amino acids were derivatized with pentafluoropropionic anhydride and pentafluoropropanol. The derivatization products were stable for several days. The technique has been applied to the assay of GABA and Glu in five amygdaloid nuclei of the rat brain. The GABA level was high in the central and medial nuclei, whereas the Glu level was high in the lateral and basal nuclei. The regional distribution of GABA was different from that of Glu within the amygdaloid nuclei.     

Use of Inhibitors of γ-Aminobutyric Acid (GABA) Transaminase for the Estimation of GABA Turnover in Various Brain Regions of Rats: A Reevaluation of Aminooxyacetic Acid

The technique of estimating gamma-aminobutyric acid (GABA) turnover by inhibiting its major degrading enzyme GABA-T (4-aminobutyrate:2-oxoglutarate aminotransferase; EC 2.6.1.19) and measuring GABA accumulation has been used repeatedly, but, at least in rats, its usefulness has been limited by several difficulties, including marked differences in the degree of GABA-T inhibition in different brain regions after systemic injection of GABA-T inhibitors. In an attempt to improve this type of approach for measuring GABA turnover, the time course of GABA-T inhibition and accumulation of GABA in 12 regions of rat brain has been studied after systemic administration of aminooxyacetic acid (AOAA), injected at various doses and with different routes of administration. A total and rapidly occurring inhibition of GABA-T in all regions was obtained with intraperitoneal injection of 100 mg/kg AOAA, whereas after lower doses, marked regional differences in the degree of GABA-T inhibition were found, thus leading to underestimation of GABA synthesis rates, e.g., in substantia nigra. The activity of the GABA-synthesizing enzyme GAD (L-glutamate-1-decarboxylase; EC 4.1.1.15) was not reduced significantly at any time after intraperitoneal injection of AOAA, except for a small decrease in olfactory bulbs. Even the highest dose of AOAA tested (100 mg/kg) was not associated with toxicity in rats, but induced motor impairment, which was obviously related to the marked GABA accumulation found with this dose. The increase in GABA concentrations induced with intraperitoneal injection of 100 mg/kg AOAA was rapid in onset, allowing one to estimate GABA turnover rates from the initial rate of GABA accumulation, i.e., during the first 30 min after AOAA injection. GABA turnover rates thus determined were correlated in a highly significant fashion with the GAD activities determined in brain regions, with highest turnover rates measured in substantia nigra, hypothalamus, olfactory bulb, and tectum. Pretreatment of rats with diazepam, 5 mg/kg i.p., 5-30 min prior to AOAA, reduced the AOAA-induced GABA accumulation in all 12 regions examined, most probably as a result of potentiation of postsynaptic GABA function. The data indicate that AOAA is a valuable tool for regional GABA turnover studies in rats, provided the GABA-T inhibitor is administered in sufficiently high doses to obtain complete inhibition of GABA degradation.     

Anticonvulsant Activity of Muscimol and γ-Aminobutyric Acid Against Pyridoxal Phosphate-Induced Epileptic Seizures

The intracerebroventricular injection of pyridoxal phosphate (PLP, 0.125-1.25 μmol/rat) causes epileptic seizures (4 min → 1 min) that are preventable or reversible by GABA (1 μmol/rat), by muscimol (O.025 μmol/rat), or by diazepam (1.75 μmol/rat). At the peak of PLP-induced convulsions, the activities of GAD and GABA-T in 14 regions of rat brain remained unaltered, whereas the concentrations of PLP remained elevated. The PLP-induced convulsion was blocked by DABA (10 μmol/rat) but was not altered by β-alanine (50 μmol/rat). The previous in vitro studies have shown that PLP increases the uptake of [³H]GABA into synaptosomes and inhibits the binding of [³H]GABA to synaptic membranes. These data suggest that PLP-induced convulsion is due to reduced availability of GABA to its recognition sites, rather than to alteration in the activity of GABA metabolizing enzymes, or unavailability of PLP as a coenzyme for GAD and GABA-T. Since the duration of PLP-induced epileptic seizures is short and can be prevented by GABA agonists, PLP may be used as a tool to study the nature of GABA-mediated neuroinhibition and the properties of GABA receptor sites.     

The γ-Aminobutyrate Content of Nerve Endings (Synaptosomes) in Mice After the Intramuscular Injection of γ-Aminobutyrate-Elevating Agents: A Possible Role in Anticonvulsant Activity

Abstract: The intramuscular administration of L-cycloserine, gabaculine, and aminooxyacetic acid caused significant, time-dependent increases in the γ-aminobutyric acid (GABA) content of both whole brain and synaptosomalenriched preparations obtained from the tissue, a linear relationship being observed between the two parameters. In contrast, the administration of hydrazine resulted in a large increase in whole brain GABA level, with little change in the synaptosomal GABA content. The key factor in these different responses appeared to be the degree of inhibition of glutamic acid decarboxylase by the drugs. Pretreatment of mice with the GABA-elevating agents resulted in a delay in the onset of seizures, which was related directly to the increase in synaptosomal GABA content. Although the seizures were delayed, they occurred while the GABA content of nerve endings (synaptosomes) was above that in preparations from untreated animals. The decrease in GABA content at the onset of seizures, expressed as a percentage of the level at the time of injection of the convulsant agent, was, however, reasonably constant. A hypothesis to explain these results is proposed.     

γ-Aminobutyric Acid Release from Synaptosomes Prepared from Rats Treated with Isonicotinic Acid Hydrazide and Gabaculine

The potassium-stimulated release of gamma-aminobutyric acid (GABA) from synaptosomes was determined in preparations from control rats and from rats treated with a convulsant agent [isonicotinic acid hydrazide (INH)] and an anticonvulsant agent (gabaculine). INH treatment brought about a significant decrease in Ca2+-dependent release of GABA with no effect on Ca2+-independent release, whereas gabaculine caused an increase in Ca2+-independent release with no effect on Ca2+-dependent release of GABA. Thus, the anticonvulsant action of gabaculine was not a simple reversal of the effects of INH on GABA release. The results indicate that there are at least two pools of GABA in nerve endings and support the hypothesis that exogenous GABA is taken up first into a pool that supplies GABA for Ca2+-independent release and then is transferred to a second pool (Ca2+-dependent releasable), where it mixes with newly synthesized GABA.