Endogenous GABA Content Research Articles

Norepinephrine turnover in the goldfish brain is modulated by sex steroids and GABA

It is known that norepinephrine (NE) is important in the neuroendocrine control of pituitary gonadotropin II (GTH-II) and growth hormone (GH) release but very little is known about the factors regulating NE neurons in the goldfish brain. Female gonad-intact goldfish were implanted intraperitoneally (100 μg/g) with testosterone (T) or estradiol (E 2) to elevate serum steroid levels. High-performance liquid chromatography measurements showed that steroid implantation had no effect on NE content in the telencephalon, including preoptic area (TEL-POA), or the hypothalamus (HYP). The turnover rate of NE was estimated from the rate of depletion of NE content from tissues following inhibition of tyrosine hydroxylase byα-methyl- p-tyrosine (240 μg/g). The present study demonstrates that E 2 can decrease NE turnover rates in TEL-POA and HYP of sexually regressed goldfish (August). The results in recrudescent fish (November), however, indicate a more complex interaction of E 2 with NE neurons since E 2 increased NE turnover in TEL-POA and HYP in these animals. Testosterone (T) has less prominent effects on NE turnover rates in TEL-POA and HYP; the only significant effect of T-implantation was a small reduction of NE turnover in the TEL-POA of sexually recrudescent fish. Elevation of endogenous brain GABA concentrations by injection of the GABA transaminase inhibitor, γ-vinyl-GABA (300 μg/g), significantly reduced NE turnover in TEL-POA. These data demonstrate that goldfish NE neurons in the TEL-POA are sensitive to regulation by changes in circulating sex steroids and by increases in brain GABA.

GABAergic Control of Receptivity in the Female Rat

GABAergic neurotransmission has been implicated in the control of the steroid-dependent behavior, lordosis. GABA has dual effects on lordosis: it facilitates lordosis through actions in the medial hypothalamus (mHYP) and it inhibits lordosis through actions in the preoptic area (POA). In the present study, gonadally intact and ovariectomized female rats were behaviorally tested with a sexually active male. Brains were removed from sexually receptive female either 1 or 24 h after behavioral testing. There was a significant difference in endogenous GABA concentration in HYP and POA between receptive, postreceptive and ovariectomized nonreceptive females. Specifically, GABA levels in postreceptive females were higher in the HYP (20%) and lower in the POA (21%) in comparison to receptive females (p less than 0.05). There was also a significant change in binding parameters of 3H-muscimol in the HYP and POA of receptive females as compared to 24 h postreceptive and ovariectomized rats. Attempts to modulate 3H-GABA release from hypothalamic tissue slices by estrogen or progesterone in ovariectomized rats yielded no effect on this parameter.

Handling habituation and ehlordiazepoxide have different effects on GABA and 5-HT function in the frontal cortex and hippocampus

In slices of frontal cortex and hippocampus from rats that had been habituated to handling for 21 days, there was significantly less 20 mM K +-evoked release of [ 14C]GABA ([ 14C]7-aminobutyric acid) compared with rats naive to handling. Handling for 21 days also significantly increased the uptake of [ 14C]GABA into frontal cortex and hippocampus. The change in uptake in the hippocampus was independent of any changes in release and could account for the apparent change in evoked release; in the cortex there were no independent changes in uptake and K +-evoked release. When the changes in uptake were taken into account, there were no independent changes in basal release of GABA in either region. HPLC analysis showed the change in uptake was not due to differences between the groups in endogenous GABA concentrations. Acute administration of chlordiazepoxide (CDP 7.5 mg/kg i.p.) to handling naive rats also significantly reduced K +-evoked [ 14C]GABA release from the cortex and hippocampus, but basal release and GABA uptake were unchanged. Neither handling nor CDP administration significantly changed the K +-evoked [ 3H]5-HT release, however the uptake of 5-HT and its basal release in both regions were both significantly and independently increased in animals habituated to handling, compared with handling naive animals. In the hippocampus, the endogenous 5-HT concentrations were significantly lower in the rats that had received 21 days of handung, compared with handling naive rats. In the cortex the endogenous 5-hydroxyindoleacetic acid concentrations were significantly lower in the group that had been handled for 21 days. Thus both the GABA and 5-HT systems were responsive to handling habituation.

Gamma-Aminobutyric acid function in the rat striatum is under the double influence of nigrostriatal dopaminergic and thalamostriatal inputs: two modes of regulation?

Glutamic acid decarboxylase (GAD), gamma-[3H]-aminobutyric acid [( 3H]GABA) high-affinity uptake into synaptosomes, and endogenous GABA content were measured in the rat striatum 2-3 weeks following 6-hydroxydopamine injection in the ipsilateral substantia nigra to destroy the nigrostriatal dopaminergic pathway and after kainic acid injection into the centromedial-parafascicular complex of the ipsilateral thalamus to lesion the thalamostriatal input. Both lesions resulted in apparent GAD increase concomitant with a decreased [3H]GABA uptake into striatal synaptosomes. GABA content was increased selectively following the dopaminergic lesion. Kinetic analysis of the uptake process for [3H]GABA showed selectively a decreased Vmax following the dopaminergic lesion; in animals with thalamic lesion, however, the change only concerned the Km, which showed a decreased affinity of the transport sites for [3H]GABA. Determination of Km and Vmax for GAD action on its substrate glutamic acid showed an increased affinity of GAD for glutamic acid in the case of the dopaminergic lesion without any change in Vmax, whereas the thalamic lesion resulted in GAD increase concomitant with a selective increase in Vmax. These data suggest that striatal GABA neurons are under the influence of nigrostriatal dopaminergic neurons which may reduce the GABA turnover, whereas the exact nature of the powerful control also revealed on these neurons following thalamic lesion remains to be determined. Both lesions induced adaptive neurochemical responses of striatal GABA neurons, possibly reflecting in the case of the dopaminergic deprivation an increased GABA turnover.

Benzodiazepines increase tonic component of postdecapitation convulsions in mice

The effects of benzodiazepines (BDZs) and GABA system on tonic and clonic component of postdecapitation convulsion (PDC) were studied in mice. Mice decapitated at the occipito-cervical junction, exerted biphasic convulsions, i.e., initially tonic and subsequently clonic convulsions. BDZs such as diazepam or clonazepam increased tonic and clonic components of PDC. These effects were not antagonized by Ro 15-1788, a benzodiazepine receptor antagonist. The increased tonic component was antagonized by the GABA receptor antagonists, bicuculline and picrotoxin, whereas the clonic component was augmented by them. Aminooxyacetic acid, which increases the endogenous GABA content by inhibiting the GABA-transaminase, increased the tonic component significantly; this increase was antagonized by both bicuculline and picrotoxin. Muscinol, a GABA agonist, however did not affect the tonic components but rather augmented the clonic component. Bicuculline and picrotoxin did not antagonize this effect of muscimol. These results indicate that endogenous GABA may play a crucial role in mediating the tonic component of PDC and the facilitation of this component by BDZs may also be due to the activation of GABA in the spinal cord. Furthermore, the mechanisms of the tonic component may be different from that of the clonic component.

Diazepam increases gamma-aminobutyric acid in human cerebrospinal fluid.

In 11 neurological patients, levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) were determined in cerebrospinal fluid (CSF) before and 1, 3, 5, and 8 min after intravenous injection of diazepam (2 or 5 mg). GABA levels increased progressively after intravenous injection of 5 but not 2 mg of the benzodiazepine, the differences from preinjection values being significant at 3, 5, and 8 min. Furthermore, when relative CSF GABA alterations determined after injection of diazepam were compared to those determined in sequential CSF aliquots of 10 patients without diazepam injection, mean GABA increases after diazepam were significantly different from controls in all CSF fractions. The data suggest that, in addition to its well-known effects on postsynaptic GABA function, diazepam may exert effects on endogenous GABA concentrations and/or on GABA release in the human CNS as reflected by elevation of GABA levels in human CSF.

Effects of GABAergic drugs on the GABA turnover in the substantia nigra and the corpus striatum of the rat.

Changes in the endogenous GABA concentration and in GABA turnover following GABA receptor stimulation or blockade were studied in the substantia nigra and the corpus striatum of the rat. The GABA agonists, muscimol, baclofen and THIP decreased the accumulation of GABA following inhibition of GABA-alpha-ketoglutaric acid aminotransferase by gamma-acetylenic GABA (GAG) in both structures investigated. Only the effect of muscimol in the substantia nigra was inhibited by the GABA antagonist, bicuculline. Muscimol, baclofen and progabide reduced the disappearance rate of GABA in the substantia nigra following inhibition of the glutamate decarboxylase by 4-deoxypyridoxine. The endogenous GABA concentration was decreased in the corpus striatum following muscimol, THIP or baclofen, probably due to a decreased synthesis of GABA. Smaller effects were seen on the endogenous GABA concentration in the substantia nigra, since both the synthesis and the utilization of GABA were decreased by muscimol and baclofen. Thus, the turnover of brain GABA might be regulated by changes in receptor activity.

Endogenous GABA concentration in cortical synaptosomes from young and aged rats

The objective of this study are threefold: (1) to compare the γ-aminobutyric acid (GABA) concentration of a preparation of cortical snaptosomes from young male rats to that of aged rats; (2) to compare the GABA concentration in synaptosomes retained by Millipore filtration for young and aged rats; and (3) to compare the GABA concentration in the synaptosomal preparation to that in synaptosomes retained by Millipore filtration for young and aged rats. The GABA concentration is measured in cortical synaptosomes from Long-Evans rats at 2 months and 30 months of age. Concentration of the synaptosomal preparation declined significantly from 2.97 mM in 2-month animals to 2.68 mM in 30-month animals. In order to compare the results with previous studies of GABA transport, in which Millipore filters are used to separate synaptosomes from incubation medium, the GABA concentration is also measured following placement of synaptosomes on 0.65μ pore size Millipore filters. For both young and aged animals, this population of synaptosomes is found to contain GABA at a concentration more than triple that in the overall population. This is the expected result if the filtration process reduce the non-synaptosomal component of the total preparation, since GABA uptake is known to be a function of the nerve-ending component. Comparison of 2-month and 30-month synaptosomes reveals that the GABA concentration of those synaptosomes retained by the filtration process declined to a greater extent in the aged group than did the content of the overall population (from 9.33mM to 8.37mM). The results are consistent with previous studies of the aging of the GABA transporter in this preparation, in which it was found that the GABA transport capacity declined in the 30-month age group.

Central GABA mechanisms during postnatal development in the rat: neurochemical characteristics.

Various biochemical characteristics of the developing GABA system was studied in rats from 1 to 60 days of age. Endogenous GABA concentrations were high in the limbic system, midbrain, brain stem and spinal cord at birth. Until 7 days of postnatal age, GABA concentrations generally decreased, thereafter an increase was seen and at 60 days of age the GABA concentrations exceeded those found in the neonate except for the spinal cord regions. After GABA-T inhibition with AOAA, GABA concentrations increased in all brain regions, however considerably more marked in the 28 days old rats compared to the 4 days old animals. Turnover rate of GABA was estimated by investigating the rate of disappearance of GABA after GAD inhibition with 3-MPA. Calculated turnover time of whole brain GABA was 34.1 min in the 4 days old rats and 19.9 min in the 28 days old animals. The results from this investigation clearly indicate a caudal to rostral maturational gradient in the development of endogenous GABA concentrations as well as synthesis capacity. Furthermore, turnover rate of total whole brain GABA but probably not of GABA in the neuronal pool is retarded in the 4 days old rats compared to the adolescent animals.

Alterations in a hypothalamic GABA system in the spontaneously hypertensive rat

The density (Bmax) of muscimol and clonazepam binding to hypothalamic membranes from spontaneously hypertensive rats (SHR) was reduced compared to age-matched Wistar Kyoto (WKY) animals in the period 80 – 120 days. There were no significant differences in dissociation constant (Kd) for either ligand at this time. At 30 – 36 days, prior to development of pronounced hypertension, there were no differences in Kd or Bmax for either ligand in SHR and WKY animals. There were also deficits in endogenous hypothalamic GABA concentrations in SHR at 75 and 120 days as compared to WKY. The hypothesis is advanced, that there may be a dysfunction of a hypothalamic GABA system in the SHR rat as hypertension develops.

The release of [3H]GABA formed from [3H]glutamate in rat hippocampal slices: comparison with endogenous and exogenous labeled GABA.

to compare the storage and release of endogenous GABA, of [3H]GABA formed endogenously from glutamate, and of exogenous [14C]GABA, hippocampal slices were incubated with 5 microCi/ml [3,4-3H]1-glutamate and 0.5 microCi/ml [U-14C]GABA and then were superfused in the presence or absence of Ca+ with either 50 mM K+ or 50 microM veratridine. Endogenous GABA was determined by high performance liquid chromatography which separated labeled GABA from its precursors and metabolites. Exogenous [14C]GABA content of the slices declined spontaneously while endogenous GABA and endogenously formed [3H]GABA stayed constant over a 48 min period. In the presence of Ca+ 50 mM K+ and in the presence or absence of Ca2+ veratridine released exogenous [14C]GABA more rapidly than endogenous or endogenously formed [3H]GABA, the release of the latter two occurring always in parallel. The initial specific activity of released exogenous [14C]GABA was three times, while that of endogenously formed [3H]GABA was only 50% higher than that in the slices. There was an excess of endogenous GABA content following superfusion with 50 mM K+ and Ca2+, which did not occur in the absence of Ca2+ or after veratridine. The observation that endogenous GABA and [3H]GABA formed endogenously from glutamate are stored and released in parallel but differently from exogenous labelled GABA, suggests that exogenous [3H] glutamate can enter a glutamate pool that normally serves as precursor of GABA.

Formula omitted] inactivation of γ-aminobutyric acid-α-ketoglutarate transaminase by 4-amino-5-fluoropentanoic acid

Intraperitoneal injection into mice of varying concentrations of (S)-4-amino-5-fluoropentanoic acid ((S)-AFPA) produces a dose-dependent irreversible decrease in brain γ-aminobutyric acid-α-ketoglutaric acid aminotransferase (E.C. 2.6.1.19) activity. Concomitant with this inactivation is an increase in whole brain γ-aminobutyric acid (GABA) levels. Four hours after a dose of 100 mg/kg body weight of (S)-AFPA to mice, endogenous brain GABA concentrations increase to 16 times that of the untreated animals and the enzyme activity decreases to 20% that of the controls. The binding of (S)-AFPA to GABA receptors was more than three orders of magnitude poorer than for GABA itself.

A steroid derivative, R 5135, antagonizes the GABA/benzodiazepine receptor interaction

A novel steroid derivative, R 5135 (3α-hydroxy-16-imino-5β-17-aza-androstan-11-one) showed high affinity for both the GABA and glycine receptors in vitro. It also displaced [ 3H]diazepam from the benzodiazepine receptor in a rat cortex membrane preparation, but in this case a plateau occurred in the displacement curve at a concentration of R 5135 between 10 −7 and 10 −6 M, where binding was decreased by about 50 %. The “high affinity” component of R 5135 inhibition no longer appeared when the endogenous GABA concentration was reduced by extensive washing of the membrane preparation and it reappeared when GABA was re-introduced. Thus R 5135 behaves as a powerful antagonist of the GABA stimulation of [ 3H]diazepam binding, being 500 times more active than the GABA-antagonist bicuculline. The dual interaction between R 5135 and GABA and glycine receptors suggests that these may share some common structural feature or that they have overlapping specificity.

Alterations in γ-aminobutyric acid content in the rat superior cervical ganglion and pineal gland

The endogenous γ-aminobutyric acid (GABA) content of the rat pineal gland and superior cervical ganglion (SCG) was measured by high pressure liquid chromatography. It was found that GABA levels in both tissues increased after decapitation of the animals. The GABA content of tissues frozen within 20 seconds after decapitation was the same as that of tissues removed from animals killed by microwave irradiation. Amino-oxyacetic acid, a GABA-transaminase inhibitor, increased the endogenous GABA content of both of these tissues. Dimethylphenylpiperizinium or isoniazid administration did not alter GABA levels in these tissues. Isoproterenol increased the GABA content of the SCG but did not change the pineal gland GABA levels. The ability of the pineal gland to take up and accumulate 3H-GABA was significantly reduced in rats that had been ganglionectomized. A fluctuation in endogenous GABA levels in the pineal gland was seen to occur when measures were taken at different times of the day. These results tend to suggest that GABA may have some functional role in the pineal gland and the superior cervical ganglion.

Effect of ethanolamine- O-sulphate on regional gaba metabolism in the mouse brain

The distribution of [ 3 H]ethanolamine-O- sulphate (EOS) and the regional changes in GABA metabolism after 160 μg (8 mg/kg) intracerebroventricularly and 2 g/kg subcutaneously have been studied in the mouse brain. Over 60 per cent of the injected radioactivity (given i.c.v.) was lost within the first hr. That remaining at 24 hr, however, after subcellular fractionation appeared to be associated with GABA-transaminase (GABA-T). The greatest binding occurred in the hypothalamus, an area with a high endogenous GABA concentration and the highest turnover rate of those areas studied. GABA turnover rates calculated over a 4 hr period for both 160 μg i.c.v. and 2 g/kg s.c. correlated (P < 0.001) with the endogenous GABA concentrations. EOS 2 g/kg s.c. was as effective as 160 μg i.c.v. in inhibiting GABA-T in the brain, but has a slower onset of action. The possibility of a larger GABA-T pool in the hypothalamus than in the other areas studied is discussed.

Effect of dark adaptation on the GABA system in retina

The levels of l-glutamate-1-car☐y-lyase (GAD), 4-aminobutyrate-2-oxoglutarate-aminotransferase (GABA-T) and free amino acids, and the active accumulation of [ 3H]GABA and [ 14C]taurine have been measured in light- and dark-adapted retinae from rat, chicken, frog and goldfish. No significant changes in any of these parameters with state of adaptation to light were evident in rat or chicken retina. However, in retinae from goldfish and frog respectively, decreases in [ 3H]GABA accumulation and GAD activity were noted in the dark. In both cases these changes were accompanied by a fall in the endogenous GABA content of the tissue, but this difference was only significant in goldfish retina.

GABA—2-oxoglutarate transaminase, glutamate decarboxylase and the half-life of GABA in different areas of rat brain

The activity of the enzymes GABA—2-oxoglutarate transaminase (GABA-T) and glutamate decarboxylase (GAD), the endogenous GABA content and the half-life of GABA have been estimated in whole rat brain, cerebellum, pons/medulla, midbrain, cerebral cortex/hippocampus, striatum and hypothalamus. The efflux of 3H-GABA in all brain areas consists of at least two components, a fast phase having a half-life of between 0.5 and 1 hr and a slower phase with a half-life exceeding 1 hr. In addition, significant differences in the calculated rates of GABA turnover among brain regions were found. Endogenous GABA and also the faster turnover phase are proportional to GAD activity but are not dependent on concomitant GABA-T activity. The possible significance of these results are discussed in relation to the postulated role of GABA as a neurotransmitter in the CNS.

Effect of ethanol on entry of some substances into the brains of rats.

We have examined the effects of a subanesthetic dose of ethanol on the entry into the brains of rats of three 14C-labelled substances which are known to affect brain function. All substances were given intraperitoneally. When ethanol was given the concentration of pentobarbital in the brain reached and was maintained at a higher level than without ethanol. The barbiturate entered the brain and became distributed between plasma, red cells, and brain extremely rapidly in the presence or absence of ethanol. Administration of ethanol decreased the rate of breakdown of pentobarbital, so that higher levels of unchanged pentobarbital were maintained in blood, liver, and brain. Breakdown of the barbiturate occurred rapidly in liver but almost no radioactive breakdown products entered or were formed in the brain. Thiamine entered the brain sparingly but, after 60 min, the ratio of radioactivity per gram of brain to that per milliliter of plasma became considerably greater with ethanol than without. In the absence or presence of ethanol this ratio increased with time due to a rapid disappearance of radioactivity from the plasma while radioactivity in the brain tended to increase slightly. In the presence of ethanol the rate of disappearance from the plasma increased, presumably due to increased uptake and metabolism of thiamine by other tissues. Urinary excretion of radioactivity decreased. Injected γ-aminobutyric acid (GABA), entered the brain very sparingly and much of what entered was metabolized. Ethanol had no effect on the endogenous GABA concentration in the brain nor on the entry or metabolism of injected GABA.