Injection Of Aminooxyacetic Acid Research Articles

Carnosine and anserine homeostasis in skeletal muscle and heart is controlled by β-alanine transamination.

Using recombinant DNA technology, the present study provides the first strong and direct evidence indicating that β-alanine is an efficient substrate for the mammalian transaminating enzymes 4-aminobutyrate-2-oxoglutarate transaminase and alanine-glyoxylate transaminase. The concentration of carnosine and anserine in murine skeletal and heart muscle depends on circulating availability of β-alanine, which is in turn controlled by degradation of β-alanine in liver and kidney. Chronic oral β-alanine supplementation is a popular ergogenic strategy in sports because it can increase the intracellular carnosine concentration and subsequently improve the performance of high-intensity exercises. The present study can partly explain why the β-alanine supplementation protocol is so inefficient, by demonstrating that exogenous β-alanine can be effectively routed toward oxidation. The metabolic fate of orally ingested β-alanine is largely unknown. Chronic β-alanine supplementation is becoming increasingly popular for improving high-intensity exercise performance because it is the rate-limiting precursor of the dipeptide carnosine (β-alanyl-l-histidine) in muscle. However, only a small fraction (3-6%) of the ingested β-alanine is used for carnosine synthesis. Thus, the present study aimed to investigate the putative contribution of two β-alanine transamination enzymes, namely 4-aminobutyrate-2-oxoglutarate transaminase (GABA-T) and alanine-glyoxylate transaminase (AGXT2), to the homeostasis of carnosine and its methylated analogue anserine. We found that, when transfected into HEK293T cells, recombinant mouse and human GABA-T and AGXT2 are able to transaminate β-alanine efficiently. The reaction catalysed by GABA-T is inhibited by vigabatrin, whereas both GABA-T and AGXT2 activity is inhibited by aminooxyacetic acid (AOA). Both GABA-T and AGXT2 are highly expressed in the mouse liver and kidney and the administration of the inhibitors effectively reduced their enzyme activity in liver (GABA-T for vigabatrin; GABA-T and AGXT2 for AOA). In vivo, injection of AOA in C57BL/6 mice placed on β-alanine (0.1% w/v in drinking water) for 2weeks lead to a 3-fold increase in circulating β-alanine levels and to significantly higher levels of carnosine and anserine in skeletal muscle and heart. By contrast, specific inhibition of GABA-T by vigabatrin did not affect carnosine and anserine levels in either tissue. Collectively, these data demonstrate that homeostasis of carnosine and anserine in mammalian skeletal muscle and heart is controlled by circulating β-alanine levels, which are suppressed by hepatic and renal β-alanine transamination upon oral β-alanine intake.

Neurons in layer III of the entorhinal cortex. A role in epileptogenesis and epilepsy?

A preferential lesion of neurons in layer III of the entorhinal cortex (EC) is often observed in patients suffering from temporal lobe epilepsy and in several animal models of the disease. This lesion is duplicated in rats by a focal, intra-entorhinal injection of the "indirect" excitotoxin aminooxyacetic acid (AOAA), providing a model that can be used to study the mechanisms underlying seizure-induced cell death and epilepsy. Doomed neurons in the EC and in several associated limbic structures show pathological changes within hours after the AOAA injection, but GABAergic neurons in layer III of the EC are quite resistant. This pattern of neuron loss eventually results in hippocampal and entorhinal hyperexcitability. Notably, the seizure-induced death of layer III neurons in the EC can be attenuated by eliminating the prominent excitatory input from the presubiculum. Taken together, these results suggest opportunities to target parahippocampal structures for the treatment of temporal lobe epilepsy.

Ultrastructure and immunocytochemical distribution of GABA in layer III of the rat medial entorhinal cortex following aminooxyacetic acid-induced seizures.

Layer III of the entorhinal cortex (EC) is lesioned in patients with temporal lobe epilepsy (TLE). A similar neuropathology is also present in different animal models of TLE. For example, injection of the "indirect" excitotoxin aminooxyacetic acid (AOAA) into the EC of rats causes behavioral seizures and preferential loss of neurons in layer III of the medial EC. The animals also develop hyperexcitability of the EC and the hippocampal region CA1. To further explore the neuropathological changes within the EC, the ultrastructure and distribution of GABA-like immunoreactivity were assessed in layer III, 28 days after an intraentorhinal AOAA injection. At this time point, light microscopic preparations revealed that a large proportion of pyramidal (putative excitatory) neurons in layer III of the medial EC had degenerated, whereas GABA-immunoreactive neurons had survived. In immunogold-labeled ultrathin sections, the lesioned neuropil was found to contain morphologically intact GABA-containing neurons and nerve terminals. Pathologically swollen dendrites and electron-dense neuronal profiles were present in the lesioned sector as well. The majority of the electron-dense profiles was identified as degenerating dendritic spines that were closely apposed to strongly glutamate-immunopositive axon terminals. Thus, the entorhinal chemoarchitecture is dramatically altered following an episode of AOAA-induced epileptic seizures. One possible consequence of this pathology is a reduced "drive" of the surviving layer III GABA neurons, which in turn may cause hyperexcitability of the EC and the hippocampus. These findings may be of relevance for the genesis and spread of temporal lobe seizures.

Neuronal damage after the injection of amino-oxyacetic acid into the rat entorhinal cortex: a silver impregnation study

In rats, most neurons in layer III of the medial entorhinal cortex are exquisitely vulnerable to prolonged seizure activity. These neurons have also been shown to die preferentially in the entorhinal cortex of patients with temporal lobe epilepsy. This lesion can be duplicated in rats by a focal injection of the indirect excitotoxin amino-oxyacetic acid into the entorhinal cortex. The present study was designed to examine the neuropathological consequences of an intra-entorhinal amino-oxyacetic acid injection at various time-points with a sensitive silver-staining method for the visualization of damaged neurons. After 3h, affected cells with prominently stained processes were readily observed in the transition zone of the hippocampal CA1 field and the subiculum, but no silver-stained neurons were seen in the entorhinal cortex. Less consistently, damaged neurons were observed in the presubiculum, in the temporal and perirhinal cortices and in the lateral amygdaloid nucleus. At 6h after an amino-oxyacetic acid injection, numerous silver-stained neurons, which were typically devoid of processes, were also seen in layer III of the medial entorhinal cortex. This pattern of neurodegeneration remained similar at 12 and 24h following the amino-oxyacetic acid injection, though many silver-stained neurons were noted in layer II of the lateral entorhinal cortex as well. Notably, at five days, silver-stained neurons had disappeared. Instead, dendritic arbors, debris of degenerated neurons and reactive glial cells were present in lesioned brain regions. These data demonstrate the chronology and the extent of neuronal damage following an intra-entorhinal injection of amino-oxyacetic acid.The results suggest that a detailed examination of the temporal sequence of neuronal death in the entorhinal cortex and in extra-entorhinal areas is likely to benefit our understanding of the pathophysiology of temporal lobe epilepsy.

Hyperexcitability of entorhinal cortex and hippocampus after application of aminooxyacetic acid (AOAA) to layer III of the rat medial entorhinal cortex in vitro.

1. Injection of aminooxyacetic acid (AOAA) into the entorhinal cortex in vivo produces acute seizures and cell loss in medial entorhinal cortex. To understand these effects, AOAA was applied directly to the medial entorhinal cortex in slices containing both the entorhinal cortex and hippocampus. Extracellular and intracellular recordings were made in both the entorhinal cortex and hippocampus to study responses to angular bundle stimulation and spontaneous activity. 2. AOAA was applied focally by leak from a micropipette or by pressure ejection. Evoked potentials increased gradually within 5 min of application, particularly the late, negative components. Evoked potentials continued to increase for up to 1 h, and these changes persisted for the remainder of the experiment (up to 5 h after drug application). 3. Paired pulse facilitation (100-ms interval) was also enhanced after AOAA application. Increasing stimulus frequency to 1-10 Hz increased evoked potentials further, and after several seconds of such stimulation multiple field potentials occurred. When stimulation was stopped at this point, repetitive field potentials occurred spontaneously for 1-2 min. These recordings, and simultaneous extracellular recordings in different layers, indicated that spontaneous synchronous activity occurred in entorhinal neurons. Intracellularly labeled cortical pyramidal cells depolarized and discharged during spontaneous and evoked field potentials. 4. The effects of AOAA were blocked reversibly by bath application of the N-methyl-D-aspartate (NMDA) receptor antagonist D-amino-5-phosphonovalerate (D-APV; 25 microM) or focal application of D-APV to the medial entorhinal cortex. 5. Simultaneous extracellular recordings from the entorhinal cortex and hippocampus demonstrated that spontaneous synchronous activity in layer III was often followed within several milliseconds by negative field potentials in the terminal zones of the perforant path (stratum moleculare of the dentate gyrus and stratum lacunosum-moleculare of area CA1). The extracellular potentials recorded in the dentate gyrus corresponded to excitatory postsynaptic potentials and action potentials in dentate granule cells. However, extracellular potentials in area CA1 were small and rarely correlated with discharge in CA1 pyramidal cells. 6. The results demonstrate that AOAA application leads to an NMDA-receptor-dependent enhancement of evoked potentials in medial entorhinal cortical neurons, which appears to be irreversible. The potentials can be facilitated by repetitive stimulation, and lead to synchronized discharges of entorhinal neurons. The discharges invade other areas such as the hippocampus, indicating how seizure activity may spread after AOAA injection in vivo. These data suggest that AOAA may be a useful tool to study longlasting changes in NMDA receptor function that lead to epileptiform activity and neurodegeneration.

The novel antiepileptic drug levetiracetam (ucb L059) induces alterations in GABA metabolism and turnover in discrete areas of rat brain and reduces neuronal activity in substantia nigra pars reticulata

Levetiracetam (( S)-α-ethyl -2-oxo-pyrrolidine acetamide, ucb L059) is a novel anticonvulsant drug presently in clinical development. Its mechanism of action is unknown although a recently reported novel specific binding site for [ 3H]levetiracetam, unique to brain, may be involved. This binding site has not yet been characterized, but some evidence suggested a possibly indirect interaction with the GABA system. We therefore examined levetiracetam's effects on GABA metabolism and turnover in several rat brain regions after systemic administration of anticonvulsant doses. Furthermore, in order to study functional effects of levetiracetam on a well defined system of GABAergic neurons in a brain region that has been critically involved in anticonvulsant drug action, we examined levetiracetam's action on spontaneous firing of substantia nigra pars reticulata (SNR) neurons in anesthetized rats. Although levetiracetam did not alter the activity of the GABA synthesizing and degrading enzymes glutamic acid decarboxylase (GAD) and GABA aminotransferase (GABA-T) in vitro, systemic administration induced significant alterations in these enzymes in several brain regions, indicating that these enzyme alterations were no direct drug effects but a consequence of postsynaptic changes in either GABAergic or other neurotransmitter-related systems. In the striatum, levetiracetam, 170 mg/kg i.p., induced a significant increase in GABA-T activity while GAD activity markedly decreased. When GABA turnover was estimated after inhibition of GABA-T by aminooxyacetic acid (AOAA), treatment with levetiracetam (given 15 min prior to injection of AOAA) significantly reduced GABA turnover in the striatum. Since the substantia nigra pars reticulata (SNR) receives a strong GABAergic input from the striatum, we examined if the alterations in GABA metabolism and turnover in the striatum led to functional alterations in neuronal activity in the SNR by recording single unit activity of SNR neurons after i.p. injection of levetiracetam. While injection of vehicle did not affect SNR neuronal activity, a significant decrease in spontaneous neuronal firing was recorded after levetiracetam. Since a substantial body of evidence suggests that the SNR is a critical site at which decrease of neuronal firing results in protection against various seizure types, the suppressive effect of levetiracetam on SNR activity may contribute to the anticonvulsant action of this drug.

Prolactin- and testosterone-induced inhibition of LH secretion after orchidectomy: role of preoptic and tuberoinfundibular γ-aminobutyric acidergic neurones

The inhibitory amino acid neurotransmitter gamma-aminobutyric acid (GABA) may play an important role in the regulation of LH-releasing hormone secretion. The present study examined the effect of prolactin on GABAergic neuronal activity in microdissected brain regions of the orchidectomized rat, to determine whether inhibition of LH secretion after castration by acute hyperprolactinaemia was associated with prolactin-induced changes in GABAergic neuronal activity. The effects of prolactin were contrasted with the effects of testosterone on hypothalamic GABAergic neurones after orchidectomy. GABA concentrations were measured by high pressure liquid chromatography in eight microdissected brain regions in untreated rats and 60 min after inhibition of the GABA catabolic enzyme GABA transaminase by injection of amino-oxyacetic acid (AOAA). The rate of GABA accumulation in microdissected brain regions following injection of AOAA was taken as an index of GABAergic neuronal activity. Rats were divided into seven experimental groups: intact controls, 2 days after castration, 2 days after castration with prolactin treatment (2.5 mg ovine prolactin injected s.c. every 12 h, starting at the time of castration), 2 days after castration with testosterone replacement (30 mm silicone elastomer implant containing crystalline testosterone), 6 days after castration, 6 days after castration with prolactin treatment, and 6 days after castration with testosterone replacement. Both 2 and 6 days after castration, plasma LH was markedly elevated above levels in intact rats, and AOAA-induced GABA accumulation was significantly decreased in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis, in the medial preoptic nucleus and in the median eminence. Hyperprolactinaemia significantly reduced LH levels 2 days but not 6 days after castration. GABAergic neuronal activity, however, was not significantly affected by prolactin at either time. Testosterone replacement blocked the postcastration elevation in plasma LH and prevented the castration-induced suppression of GABAergic neuronal activity both 2 and 6 days after castration. There were no castration- or hormone-induced changes in GABAergic neurones observed in the medial or lateral septum, caudate nucleus, cingulate cortex or arcuate nucleus. These results demonstrate that the activity of GABAergic neurones terminating in the rostral hypothalamus and the median eminence is positively regulated by testosterone, and that these steroid-sensitive GABAergic neurones may be important in the negative-feedback control of LH secretion. Inhibition of LH secretion by hyperprolactinaemia, however, may not be mediated by changes in GABAergic neuronal activity.

Castration-induced decrease in the activity of medial preoptic and tuberoinfundibular GABAergic neurons is prevented by testosterone.

We recently determined that castration specifically decreased GABA turnover in discrete rostral and mediobasal hypothalamic structures. This study aimed to investigate whether testosterone could stimulate GABAergic neuronal activity in these hypothalamic GABAergic neurons in the castrate rat, and to compare the effects of episodic testosterone replacement with the constant levels provided by subcutaneous testosterone implants. Animals were divided into 4 experimental groups: intact, 48 h castrate, 48 h castrate+testosterone capsules (2 x 30 mm Silastic implants, 1.57 mm ID, 3.18 mm OD) and 48 h castrate+testosterone injections (100 micrograms/injection s.c., every 8 h). GABA concentrations were measured in 4 microdissected brain regions either before or 60 min after inhibition of the GABA degrading enzyme, GABA transaminase, by injection of aminooxyacetic acid (AOAA, 100 mg/kg i.p.). The rate of GABA accumulation in the tissue following injection of AOAA was used as an index of GABAergic neuronal activity. Castration resulted in a 10-fold increase in serum LH concentrations compared with intact rats. Either mode of testosterone administration completely prevented this castration-induced LH rise. In the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis, the medial preoptic nucleus and in the median eminence, GABA turnover was significantly reduced by castration to approximately 50% that of intact rats. Either testosterone implants or testosterone injections prevented this castration-induced decrease in GABA turnover, such that the turnover rates were not significantly different from intact rats. There was no effect of castration with or without testosterone replacement in the cingulate cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminooxyacetic acid produces excitotoxic brain injury in neonatal rats

Impairment of cellular energy metabolism plays an important role in the expression of brain injury resulting from a variety of acute neurologic disorders. The role of mitochondrial energy metabolism in excitotoxic perinatal brain injury was assessed by studying the toxicity of aminooxyacetic acid (AOAA), an inhibitor of mitochondrial malate-aspartate shunt, in postnatal (PND) 7 rats. Intrastriatal injection of AOAA produced seizures and dose-dependent excitotoxic injury. The neuronal damage was attenuated by pyridoxine suggesting involvement of pyridoxal dependent mechanisms. The lesion was selectively blocked by the NMDA antagonist MK-801 but not the AMPA antagonist GYKI-52466. Furthermore, AOAA potentiated NMDA, but not AMPA or 1S,3R-ACPD, induced brain injury. The data suggest that regional impairmeent of cellular energy metabolism is an important determinant of selective vulnerability to excitotoxic injury in perinatal rats. Furthermore, the role of impaired energy metabolism is particularly relevant to NMDA receptor mediated brain injury.

Aminooxyacetic acid causes selective neuronal loss in layer III of the rat medial entorhinal cortex

Aminooxyacetic acid (AOAA) was used to produce a selective lesion in the rat entorhinal cortex (EC). As assessed 7 days following the injection of AOAA (75 μg0.75 μl) into the EC, neuronal loss in layer III of the medial EC, particularly in its ventral portion, was consistently observed in Nissl-stained horizontal sections. This selective neurodegeneration was seen even when AOAA was injected laterally or in deeper layers. Behavioral seizures occurred between 2 and 4 h after the AOAA injection. AOAA-induced EC lesions may provide experimental models for the study of human diseases in which the EC, particularly layer III neurons, is involved.

The immediate consequences of middle cerebral artery occlusion on GABA synthesis in mouse cortex and cerebellum

The effect on γ-aminobutyric acid (GABA) synthesis of focal ischaemia in the right cortex of the mouse was investigated by performing a right middle cerebral artery (MCA) occlusion. Synthesis of GABA was determined by measurement of the rate of GABA accumulation in tissue following injection of amino oxyacetic acid (AOAA; 30 mg/kg, i.p.). Five min following the MCA occlusion, the rate of GABA synthesis in the right (ischaemic) cortex was decreased by approximately 70% compared to either the left cortex or the right cortex of untreated controls. The basal GABA concentration was however unaffected. Four hours after the occlusion the rate of GABA synthesis was similar in the right and left cortex. The rate of GABA accumulation in the cerebellum was unchanged at both times after the right MCA occlusion compared with untreated control mice. The data suggest that there is a rapid but short lasting decrease in GABA synthesis following an ischaemic insult and it is suggested that this might be associated with the EEG spiking activity that occurs at this time.

Aminooxyacetic acid produces excitotoxic lesions in the rat striatum

The neuropathological, biochemical, and behavioral effects of intrastriatal injection of aminooxyacetic acid (AOAA), a non-specific transaminase inhibitor, were examined in rats. AOAA, 0.1-1 mumol, produced neuronal damage when injected into the striatum of adult rats but failed to damage the striatum of 6-d-old or decorticated rats. AOAA-induced (0.25 mumol-1 mumol) striatal lesions in adult rats displayed excitotoxic characteristics and could be prevented by the N-methyl-D-aspartate (NMDA) receptor antagonists (-)-2-amino-7-phosphono-heptanoate (AP7; 0.25 mumol) or kynurenate (KYNA; 0.5 mumol), but not by the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX; 0.25 mumol). AOAA produced a dose-dependent reduction in striatal L-glutamate decarboxylase activity, as measured 14 d following intrastriatal injection, which could also be prevented by AP7 or KYNA, but not by NBQX. These findings suggest that AOAA-induced lesions are preferentially mediated by activation of the NMDA subtype of excitatory amino acid receptors. Behavioral studies revealed that the cataleptic response to haloperidol, 2 mg/kg, was decreased whereas the cataleptic response to arecoline, 15 mg/kg, and morphine, 15 mg/kg, was potentiated in AOAA lesioned animals 14 d following bilateral intrastriatal injections of AOAA, 0.25 and 1 mumol. In rats which received unilateral intrastriatal injection of AOAA, 0.1-1 mumol, apomorphine, 0.5 mg/kg, induced circling towards the lesioned side. Rats which received AP7, 0.25 mumol, or KYNA, 0.5 mumol, coadministered with AOAA, 0.25 mumol, behaved as vehicle-treated controls, while those which received NBQX, 0.25 mumol, and AOAA, 0.25 mumol, had behavioral patterns similar to those subjected to AOAA alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminooxyacetic Acid Results in Excitotoxin Lesions by a Novel Indirect Mechanism

Aminooxyacetic acid (AOAA) is an inhibitor of several pyridoxal phosphate-depedent enzymes in the brain. In the present experiments intrastriatal injections of AOAA produced dose-dependent excitotoxic lesions. The lesions were dependent on a pyridoxal phosphate mechanisms because pyridoxine blocked them. The lesions were blocked by the noncompetitive N-methyl-D-aspartate (NMDA) antagonist MK-801 and by coinjection of kynurenate, a result indicating an NMDA receptor-mediated excitotoxic process. Electrophysiologic studies showed that AOAA does not directly activate ligand-gated ion channels in cultured cortical or striatal neurons. Pentobarbital anesthesia attenuated the lesions. AOAA injections resulted in significant increases in lactate content and depletions of ATP levels. AOAA striatal lesions closely resemble Huntington's disease both neurochemically and histologically because they show striking sparing of NADPH-diaphorase and large neurons within the lesioned area. AOAA produces excitotoxic lesions by a novel indirect mechanism, which appears to be due to impairment of intracellular energy metabolism, secondary to its ability to block the mitochondrial malate-aspartate shunt. These results raise the possibility that a regional impairment of intracellular energy metabolism may secondarily result in excitotoxic neuronal death in chronic neurodegenerative illnesses, such as Huntington's disease.

Gabapentin increases aminooxyacetic acid-induced GABA accumulation in several regions of rat brain

Gabapentin exerts anticonvulsant effects in different animal models of seizure states and in epileptic patients with different seizure types, but the mechanism of action of these effects is unknown. In the present study, the gamma-aminobutyric acid (GABA) accumulation induced by aminooxyacetic acid (AOAA) was used as a method to study the effects of gabapentin on regional turnover of GABA in the rat brain. Gabapentin was administered at a dose of 23 mg/kg i.p. (the ED95 against tonic electroconvulsions in rats) 1, 2 and 8 h prior to injection of AOAA, 100 mg/kg, i.p. Gabapentin significantly increased the AOAA-induced GABA accumulation in most of the 12 brain regions examined, but the time course of the increases in GABA accumulation differed from region to region. Regions in which the time course of the increase in GABA accumulation was similar to the anticonvulsant time course of gabapentin included substantia nigra, amygdala and thalamus. The data suggest that an effect of gabapentin on GABA synthesis might be involved in its mechanism of anticonvulsant action.

Valproate enhances GABA turnover in the substantia nigra

The effects of the antiepileptic drug, valproate (VPA), on regional turnover of γ-aminobutyric acid (GABA) in the rat brain were studied by determining the rate of GABA accumulation following complete inhibition of GABA degradation by aminooxyacetic acid (AOAA). VPA was administered at a dose of 200 mg/kg 5, 15 and 30 min prior to injection of AOAA, 100 mg/kg. In most of the 12 regions examined, VPA did not alter the AOAA-induced GABA accumulation. However, significant increases in GABA accumulation were found in corpus striatum and, more marked, in substantia nigra. Since the substantia nigra has been identified as a substrate for the anticonvulsant action of GABAergic drugs, the data may indicate that the effect of VPA on GABA synthesis rate in this region may be involved in its mechanism of anticonvulsant action.

Inhibition of the rate of GABA synthesis in regions of rat brain following a convulsion.

1 The rate of synthesis of gamma-aminobutyric acid (GABA) in the cortex, hippocampus and striatum of rat brain was assessed by measuring the linear rate of accumulation of GABA following injection of amino-oxyacetic acid (AOAA). 2 Five min after a single electrically induced seizure there was a rise in GABA content in these brain regions and an almost total inhibition of the rate of synthesis. 3 Five min after seizure induced by the inhalant convulsant flurothyl there was no rise in GABA content in these brain regions but a similar marked degree of inhibition of GABA synthesis. 4 Two hours after the convulsion the rate of GABA synthesis had returned to control values in all three brain regions. 5 A single convulsion did not alter the glutamic acid decarboxylase activity in these brain regions either in the absence or presence of added co-factor (pyridoxal phosphate). 6 Evidence for an inhibition of GABA release following a convulsion which may be associated with the inhibition of GABA synthesis is presented in the following paper.

Relationship between drug-induced increases of GABA levels in discrete brain areas and different pharmacological effects in rats

Following the administration of two γ-aminobutyric acid-(GABA) elevating drugs, namely aminooxyacetic acid (AOAA) and valproic acid (VPA), in rats, the relationship between the magnitude and the time course of increases in GABA levels of 11 brain regions and a number of pharmacological effects was studied. AOAA (30 mg/kg i.p.) caused significant GABA increases in all brain areas but the degree and time course of these increases showed considerable variation from region to region. The most marked effects were seen in the olfactory bulb, frontal cortex and hippocampus, in which maximum GABA elevations of 100–200% were reached 4–6 hr after AOAA injection. In all the other regions studied (corpus striatum, thalamus, hypothalamus, superior and inferior colliculus, substantia nigra, pons, medulla, cerebellum), increases in GABA were less marked and, at least in part, maximum increases (30–60% over control) were already reached by 1–2 hr. In contrast to AOAA, VPA (200 mg/kg i.p.) produced significant increases in GABA levels only in the cortex, olfactory bulb, corpus striatum, hypothalamus and cerebellum, maximum effects (15–35%) being already reached 5–30 min after VPA administration. As regards pharmacological effects, AOAA caused marked hypothermia, which was maximal by 1 hr and could be reversed by increasing ambient temperature, whereas effects of VPA on body temperature were only moderate. On the other hand, both drugs exerted an almost equal, pronounced antinociceptive effect in the hot plate test. Anticonvulsant efficacy was evaluated in three seizure models, namely the maximal (tonic extension) electroconvulsive threshold, and seizures induced by pentylenetetrazol and 3-mercaptopropionic acid. Anticonvulsant effects of AOAA against electroshock and pentylenetetrazol could only be determined 1 hr after injection, at which time AOAA was inactive against 3-mercaptopropionic acid-induced seizures. VPA proved to be clearly superior to AOAA in both anticonvulsant potency and duration of action. The marked differences in functional effects between VPA and AOAA could not be related to their differential effects on GABA levels in discrete brain regions. The data thus suggest that measurement of total GABA in brain regions without consideration of the compartmentalization of the neurotransmitter is only of limited value to use in an attempt to correlate elevation of GABA levels and pharmacological effects.

On the importance of GABA-ergic neurons for the AOAA induced accumulation of GABA in the rat brain.

The accumulation of GABA induced by an intravenous injection of aminooxyacetic acid (AOAA) was followed for 1 h in five parts of the rat brain, i.e. cerebellum, medulla oblongata-pons, striatum, ventral mesencephalon and hypothalamus. The accumulation was similar in all brain parts studied when expressed as percentual increase from the basal value; an initial rapid accumulation indicating a mean turnovertime of 12--16 min during the first 5 min was gradually decreased to turnovertime of about 1--3 h during the last 30 min of observation. In order experiments brains were transected unilaterally between the substantia nigra and the striatum. Six days after the hemisection the GABA concentration of the substantia nigra of the transected side had decreased to less than 30% of the control side. The AOAA induced accumulation of GABA in the substantia nigra of the transected side was less pronounced than that of the control side. The initial rapid accumulation seen in all brain parts studied was completely lacking in the substantia nigra of the transected side. In the striatum, the transection did neither alter the GABA concentration nor the AOAA induced accumulation of GABA. As the initial rapid accumulation of GABA disappears after degeneration of GABA-ergic neurons, it is suggested that this initial phase of GABA accumulation produced by an intravenous injection of AOAA probably is the result of GABA accumulation in neurons.

Effect of aminooxyacetic acid (AOAA) on GABA levels in some parts of the rat brain.

The accumulation of GABA in the cerebellum and medulla oblongata-pons of rats has been studied after inhibition of GABA-T (EC 2.6.1.19) by different doses of AOAA. It was found that intraperitoneal (i.p.) injections of AOAA were, at least during the first hour after injection, much less effective than intravenous (i.v.) injections probably due to poor absorption i.p. After i.v. injection, AOAA caused a maximal accumulation of GABA in the cerebellum at a dose of 50 mg/kg. This maximal effect was virtually unchanged up to a dose of 150 mg/kg (the highest dose tested i.v.). If GAD (EC 4.1.1.15) was inhibited by 3-mercaptopropionic acid 30 min after AOAA (90 mg/kg i.v.) the GABA level was stable for at least another 30 min. The rate of GABA accumulation in the cerebellum during the first 15 min after AOAA (50-150 mg/kg i.v.) was 0.086 mumol/g/min and thereafter 0.034 mumol/g/min. It is concluded that AOAA in vivo in a wide dose range inhibits GABA-T almost 100% without affecting GAD to any great extent, and that the onset of action is rapid after i.v. but not after i.p. injection.

Evaluation of increases in nerve terminal-dependent vs nerve terminal-independent compartments of GABA in vivo

Evaluation of increases in nerve terminal-dependent and nerve terminal-independent compartments of GABA in vivo. Changes in GABA occurring in the absence and in the presence of GABAergic nerve terminals were estimated using rats in which the GABAergic projection to the substantia nigra (SN) was destroyed on one side of the brain. The net increase in GABA content of the GABA-denervated SN was compared with that of the intact SN after injection of amino-oxyacetic acid (AOAA), n-dipropylacetate (DPA) and γ-vinyl GABA (GVG). One week post-operative, GABA concentration in the SN on the transected hemisphere was 10–20% of control. AOAA (30 mg/kg) produced a two-fold increase in GABA content in the denervated SN, whereas DPA (300 mg/kg) was without effect. Since DPA caused an increase in GABA in the intact SN which was similar to that caused by AOAA (25–30%), it appears that the DPA induced elevation of GABA depends upon the presence of GABAergic nerve terminals whereas AOAA primarily elevates GABA in non-nerve-terminal compartments. GVG, an irreversible GABA-T inhibitor, increased GABA levels in both compartments. The predominant effect of GVG was on nerve terminal-independent GABA (4-fold increase after 900 mg/kg); the GABA increase associated with nerve terminals was smaller (30%) and did not appear until 60 hr after a single injection of GVG. The ability of DPA, AOAA and GVG to protect against chemically- and electrically-induced seizures was directly correlated with increases in nerve-terminal GABA, and not related to increases in other GABA pools.