Evaluation of the mechanisms by which gamma-amino-butyric acid in association with phosphatidylserine exerts an antiepileptic effect in the rat

The i.p. injection in rats of GABA (740 mg/Kg) after sonication with an equal amount of phosphatidylserine (PS) has an antiepileptic effect. The injection of plain GABA has no such an effect. Blood, brain and synaptosomal accumulation of exogenous labeled GABA under the two circumstances are evaluated. In the case of GABA/PS injection there is a higher passage of the exogenous labeled neurotransmitter into the blood and brain nerve endings (synaptosomes). A higher synaptosomal accumulation of the exogenous labeled neurotransmitter is found even when GABA and PS are injected separately. Since these accumulation increases occur at a time when there is the antiepileptic effect, they seem relevant to it. Our interpretation of the chain of the events resulting in the antiepileptic action is that the phospholipid facilitates from the beginning the first passage of the exogenous neurotransmitter form the peritoneum to the blood. Then a higher passage to the brain tissue and eventually to the GABA-ergic nerve endings ensues. The brisker accumulation of the exogenous neurotransmitter in the nerve endings could be at the basis of a more efficient GABA-ergic inhibitory control in the brain.     

ON THE TURNOVER OF ACETYLCHOLINE IN NERVE ENDINGS OF MOUSE BRAIN IN VIVO

Abstract— Acetylcholine is synthesized and stored in the nerve endings from which the liberation of the nerve transmittor is regulated by the nerve activity. The aim of the present investigation was to measure the in vivo turnover of acetylcholine in this subcellular acetylcholine pool. This has been carried out by injecting labelled choline intravenously and then by measuring at different time intervals the ratio between labelled choline and acetylcholine in the fractions obtained after subcellular fractionation. It was found that the ratio radioactive choline to radioactive acetylcholine was the same (2:1) in whole brain and in the nerve ending fraction 2 to 20 min after injection. Since it was assumed that the same ratio is true also for the endogenous compounds the choline pool in the nerve terminals was considered to make up 13 nmoles/g brain. The results also indicate that plasma choline is rapidly equilibrated with the nerve terminals and transformed to acetylcholine at a rate of about 5 nmoles/g brain/min.     

Association of putrescine,spermidine, spermine,and GABA with structural elements of brain cells

The present experiments are the first survey of the association of endogenous and exogenous putrescine, spermidine, and spermine with subcellular structures of rat brain cortex. The differences of distribution in subfractions obtained from salt-free and salt-containing density gradients were studied, with the following results: (1) In contrast with liver preparation, putrescine and the polyamines spermidine and spermine are not distributed in parallel with RNA. (2) In salt-containing media, putrescine and the polyamines were preferentially associated with synaptosomes and with synaptosomal membranes. Significant association with myelin constituents was observed only in salt-free media. (3) Exogenous putrescine and the polyamines were less firmly attached to synaptosomes and to synaptosomal membrane fractions than the endogenous amines. There is good evidence for similar subcellular localizations of putrescine and GABA. Putrescine seems to be entrapped in the nerve endings. (4) Uptake studies with crude mitochondria under conditions of high-affinity uptake showed no temperature-sensitive component of polyamine accumulation in synaptosomes, in contrast with GABA, monoacetylputrescine, and ornithine. (5) Polyamines bound to myelin constituents or mitochondria could be displaced by a 200-fold concentration of nonradioactive amines; this was not the case with polyamines bound to synaptosomes. Mg²⁺ did not effectively compete with spermine for binding sites at synaptic regions. (6) Electrical stimulation and stimulation by mono- and bivalent cations did not change the concentrations of the polyamines and GABA in guinea pig cortex. (7) There is no evidence for a neurotransmitter role of putrescine, spermidine, or spermine, although these compounds might function as modulators of neurotransmission.     

Studies on the release of exogenous and endogenous GABA and glutamate from rat brain synaptosomes

The aim of the present study was to determine whether exogenous radioactive GABA and glutamate previously taken up by rat brain synaptosomes are released preferentially with respect to the endogenous unlabeled amino acids. Preferential release was monitored by comparing the specific radioactivity of the amino acids released to that present in synaptosomes at the beginning and at the end of the release period. The GABA released spontaneously or by depolarizing the synaptosomes with high K⁺ in the presence of Ca²⁺ had the same specific radio-activity as that present in synaptosomes before or after superfusion. Depolarization with veratridine or superfusion with OH-GABA caused a moderate increase (15–20%) in the specific radioactivity of the GABA released and a corresponding slight decrease in that of superfused synaptosomes. In conditions causing a supraadditive release of exogenous and endogenous GABA (see ref. 13), the specific radioactivity of the GABA released was increased 20–30%. The GABA with higher-than-average specific radioactivity is probably representative of the cytoplasmic pool of this amino acid. The glutamate released spontaneously had a specific radioactivity lower than that present in synaptosomes at the start of superfusion, and also the specific radioactivity in superfused synaptosomes was lower than at the start of superfusion. The glutamate released by aspartate (by heteroexchange), by veratridine, or by high K⁺ had a specific radioactivity higher than that of the amino acid released spontaneously, similar to that present in synaptosomes at the start of superfusion, and higher than that found in superfused synaptosomes. These findings suggest that exogenous radioactive glutamate is released preferentially with respect to the endogenous amino acid and to the glutamate synthesized from glucose during the superfusion period.     

Peroxidation induced changes in synaptosomal transport of dopamine and gamma-aminobutyric acid

The effects of iron-dependent peroxidation on respiration and neurotransmitter transport of brain nerve endings has been studied. Rat brain synaptosomes were peroxidized by exposure to an ADP-Fe/ascorbate system and the protective effect of added Se, Cd, or Zn was investigated with regard to dopamine and gamma-aminobutyric acid (GABA) transport. Peroxidation impaired the respiration of synaptosomes by about 20% and caused a marked increase in dopamine uptake; but in contrast, peroxidation induced a large decrease in synaptosomal uptake of GABA. The increased dopamine transport into synaptosomes was partially prevented by the presence of Zn, Se, or Cd. The presence of Zn, Cd, or Se, in order of decreasing effectiveness, also slowed down ADP-Fe/ascorbate mediated peroxidation of synaptosomes. Peroxidation caused a significant inhibition of veratridine-dependent release of both dopamine and GABA from synaptosomes, but the KCl-dependent release of these neurotransmitters was not effected by peroxidation. These results implicate that peroxidation damage of nerve endings may lead to large changes in neurotransmitter transport thus resulting in an alteration in the function of the central nervous system.     

Development of a synaptosomal model to determine drug-induced in vivo changes in GABA-levels of nerve endings in 11 brain regions of the rat

An experimental procedure was developed which allowed the simultaneous measurement of GABA in synaptosomes from 11 regions of one rat brain. Synaptosomal fractions were prepared by conventional subcellular fractionation procedures and characterized by electron microscopy. Post-mortem increases of GABA during removal and dissection of brain tissue, homogenization and fractionation procedures could be sufficiently minimized by rapid processing of the tissue at low temperatures and inclusion of 3-mercaptopropionic acid (1 mM) in the homogenizing medium. Experiments with addition of aminooxyacetic acid (AOOA, 1 mM) to the homogenizing medium indicated that GABA was not being degraded during synaptosome preparation. The presence of exogenous GABA (1 mM) did not alter the GABA levels in the organelles, indicating that no significant redistribution of GABA occurred during subcellular fractionation. On the basis of these findings, it was suggested that synaptosomal fractions could be used as a model to monitor indirectly the drug-induced changes in GABA levels of nerve endings in discrete brain areas of the intact animal. In vivo experiments with AOAA (30 mg/kg i.p.) and valproic acid (VPA, 200 mg/kg i.p.) showed that both drugs caused differential effects on synaptosmal GABA levels in different brain regions. Although AOAA was more potent than VPA in increasing GABA in whole tissue of most brain regions, significant increases of synaptosomal GABA levels after AOAA were only determined in olfactory bulbs and frontal cerebral cortex. In contrast, VPA induced significant synaptosomal GABA increases in olfactory bulbs, hypothalamus, superior and inferior colliculus, substantia nigra, and cerebellum. The data indicate that the synaptosomal model can provide useful information on the in vivo effects of drugs on GABA levels in nerve terminals and their ability to exert this effect in specific brain areas.     

Effect of Nitric Oxide Donors on GABA Uptake by Rat Brain Synaptosomes

The effect of nitric oxide donors and L-arginine on the uptake of GABA was studied in synaptosomes purified from rat brain. The neurotransmitter uptake was significantly reduced by S-nitrosoacetylpenicillamine and by sodium nitroprusside, although in this case to a lesser extent. A slight inhibitory effect was found preincubating rat brain synaptosomes with 1 mM L-arginine as well. The S-nitrosoacetylpenicillamine effect gradually disappeared with decomposition of the substance by exposure to light. The nitric oxide effect appears to be mainly due to a decrease in the V for synaptosomal GABA uptake and seems to be related to a partial collapse of nerve endings ionic gradients. Functionally, it could result over time in a reduced availability of GABA at the synapses involved.     

Intracerebral injection of gamma vinyl GABA: Method for measuring rates of GABA synthesis in specific brain regions invivo

In order to obtain an index of the rate of GABA synthesis in different rat brain regions, we examined the rate of accumulation of GABA after irreversible inhibition of GABA-transaminase. Gamma-vinyl-GABA (GVG), a catalytic inhibitor of GABA-transaminase, was microinjected directly into each of four brain areas: superior colliculus (SC), substantia nigra (SN), frontal cortex (CTX) and caudate-putamen (CP). The subsequent rate of GABA accumulation was linear for at least 90 min in all regions, and was found to be 2–3 times higher in the SC and SN than in the CTX and CP. The nerve terminal contribution to the initial rate of GABA accumulation after GVG was determined by comparing values obtained in the intact SN with those obtained in the SN in which the GABAergic afferent terminals had been destroyed. The initial rate of GABA accumulation in the denervated SN was less than one-half of that measured in the intact SN, indicating that, under normal conditions, both nerve-terminal and non-nerve-terminal (perikarya, glia) compartments contribute to the rate of GABA accumulation after GABA-transaminase inhibition. Our results indicate that the intracerebral injection of GVG is a sensitive and reliable method for studying $\text{in}\text{vivo}$ GABA synthesis in brain. Although the rate of GABA accumulation after GVG is sensitive to changes in the nerve terminal compartment, other GABA compartments may also influence these measurements.     

EFFECTS OF 6-HYDROXYDOPAMINE ON CATECHOLAMINE CONTAINING NEURONES IN THE RAT BRAIN

After the intraventricular injection of 6-hydroxydopamine (6-OHDA), there was a long lasting reduction in the brain concentrations of noradrenaline (NA) and dopamine (DA). The brain concentration of NA was affected by lower doses of 6-OHDA than were required to deplete DA. A high dose of 6-OHDA which depleted the brain of NA and DA by 81 per cent and 66 per cent respectively, had no significant effect on brain concentrations of 5-hydroxytryptamine (5-HT) or γ-aminobutyric acid (GABA). The fall in catecholamines was accompanied by a long lasting reduction in the activities of tyrosine hydroxylase and DOPA decarboxylase in the hypothalamus and striatum, areas in the brain which are rich in catecholamine containing nerve endings. There was, however, no consistent effect on catechol-O-methyl transferase or monamine oxidase activity in these brain regions. The initial accumulation of [³H]NA into slices of the hypothalamus and striatum was markedly reduced 22–30 days after 6-OHDA treatment. These results are consistent with the evidence in the peripheral sympathetic nervous system that 6-OHDA causes a selective destruction of adrenergic nerve endings and suggest that this compound may have a similar destructive effect on catecholamine neurones in the CNS.     

Possible occurrence of ornithine-ω-aminotransferase in gabaergic neurons

The specific precursors for neurotransmitter pools of glutamate giving rise to GABA in GABAergic neurons and nerve endings have not been clearly established. Glutamate is the immediate precursor for the production of GABA and it is suggested that ornithine (from arginine) might be serving as one of the precursors of glutamate for the formation of neurotransmitter pool of GABA. Damage to GABAergic neurons in different regions of the brain in anoxia is well known. If arginine and ornithine act as precursors for GABA in GABAergic neurons, a decrease in the activities of arginase and ornithine--transferase (Orn-T) is possible in areas having the lesions involving the GABAergic neurons due to anoxia. Estimation of Orn-T and arginase in different regions of the brain of rats exposed to anoxia revealed such a possiblity.     

A possible interaction between CCKergic and GABAergic systems in the rat brain

Cholecystokinin sulfated octapeptide (CCK-8S) was given to rats i.p. at single doses of 10 and 100 nmol/kg, respectively. It produced a modification in GABA levels in several areas of the rat brain. After 30 min of injection, the lower dose (10 nmol/kg) increased GABA levels in striatum by 31% (P<0.05). The higher dose (100 nmol/kg) enhanced GABA levels either in hippocampus by 78% (P<0.05) or in frontal cerebral cortex by 81% (P<0.05) and decreased in olfactory bulbs by 57% (P<0.01). Thus, these results show that systemic injection of CCK-8S, produced regional specific changes on GABA levels in brain, and these effects were dose-dependent. Systemic pretreatment with the CCK(B) receptor antagonist, PD 135,158, 1 mg/kg i.p., on the endogenous levels of GABA in certain regions was also studied. The selective CCK(B) receptor antagonist, PD 135,158, did not have an effect per se on the endogenous levels of GABA but prevents the action induced by the neuropeptide. We suggest that the action of CCK may be mediated via a selective action on the CCK(B) receptor subtypes.     

Acute and long-lasting effects of peripheral injection of caerulein and CCK-8 on the central GABAergic system in mice

Acute and long-lasting effects of peripheral injection of caerulein (CLN) and cholecystokinin octapeptide (CCK-8) on the gamma-aminobutylic acid (GABA) content and the GABA accumulation by aminooxyacetic acid (AOAA) in the discrete brain regions of mice were examined. The content and accumulation of GABA in the striatum, hypothalamus, and frontal cortex was measured with high performance liquid chromatography with electrochemical detection (HPLC-ECD). The GABA content slightly decreased in the striatum 60 min after CLN and CCK-8 were administered, whereas it slightly increased in the hypothalamus and frontal cortex. Moreover, with CLN and CCK-8, the GABA accumulation after AOAA treatment decreased in the striatum and hypothalamus 30 min after injection. Meanwhile, when administering CLN, the GABA content as well as the GABA accumulation after AOAA treatment increased in the striatum and frontal cortex 1 day after injection, and continued to increase the second and third day in the striatum. These results showed that peripheral injection of CLN and CCK-8 had effects on the central GABAergic system with local specific actions, and also the long-lasting and time-dependent biphasic effects of CLN.     

The use of 3H-gamma-aminobutyric acid for transport studies with isolated nerve-terminals from rat brain

Isolated synaptosomes were used to study the problem of net accumulation of neurotransmitters. The time-course and the kinetics of exogenous and endogenous GABA transport were studied by liquid-scintillation counting and HPLC-amino acid analysis respectively. Different pools of GABA were suggested by a 6-fold difference in tissue-to-medium-ratio of endogenous vs. exogenous GABA. Net accumulation, exchange and net efflux of GABA was found to be a function of the GABA concentration in the incubation medium. The Kms for net accumulation and for 3H-GABA accumulation were 2.68 +/- 1.16 and 6.19 +/- 1.26 microM respectively, whereas the Vmaxs were 5.9 +/- 4.9 and 134 +/- 13 pmol/mg w.w. min respectively. This means that the transport studies which use exogenous substances (e.g. 3H-GABA) considerably overestimate the transport by overlooking the magnitude of the counter transport.     

Axonal Transport, Deposition, and Metabolic Turnover of Glycoproteins in the Rat Optic Pathway

Abstract: Following intraocular injection of [³H]fucose in the rat, radioactive glycoproteins are rapidly transported to the nerve terminals in at least two waves, one with a peak at 8 h and a second with a peak at about a week. The molecular weight distribution of radioactive peptides in ach transport wave as determined by gel electrophoresis in buffers containing sodium dodecyl sulfate is very similar. Most of the many glycopeptides in the first wave of rapid transport pass through the optic tract in unison (apparent half-life of about 15 h) and are preferentially destined for the nerve endings. However, two proteins of apparent M. W. 28,000 and 49,000 are preferentially retained in the axons. The remaining proteins, after reaching the nerve endings (superior colliculus), decay with apparent half-lives ranging from 17 to 34 h. During the second wave a large amount of the 28,000 and 49,000 M. W. peptides are again preferentially retained in the axons. The remaining proteins, on reaching the nerve endings, decay with apparent half-lives ranging from 5 to 9 days. Subcellular fractionation of the superior colliculus supports the hypothesis that the 49,000 and 28,000 M. W. peptides are the predominantly labeled glycoproteins present in myelinated axons (representing over 50% of the radioactive glycoproteins 7 days following injection), although they are probably also present in membranes of the nerve endings. A comparison with glycoprotein transport in other tracts (geniculocortical and nigrostriatal tracts) suggests that glycoprotein transport in these pathways has many similarities to glycoprotein transport in the retinal ganglion cells, and that the optic system is a good general model for axonal transport in the CNS.     

Apomorphine: sterotyped behavior and regional distribution in rat brain.

The distribution of apomorphine following subcutaneous injection of 20 mg/kg (as the hydrochloride) was measured spectrofluorometrically in specific regions of rat brain. Measurable concentrations were found in the brain within a few minutes of injection, the drug was still detectable for at least 60 min in all regions, and maximum concentration was observed 20 min after injection. Stereotyped behavior, characteristic of apomorphine action, followed a time course parallel to accumulation of the drug in the brain.     

Effect of aminooxyacetic acid on the release of preloaded [3H]GABA and radioactive metabolites from slices of developing mouse brain

The release of [³H]-aminobutyric acid (GABA) and its radioactive metabolites from slices of the cerebral cortex, cerebellum, striatum and brain stem of developing and adult mice was studied. The slices were incubated and superfused in the absence and presence of the GABA aminotransferase (GABA-T) inhibitor aminooxyacetic acid (AOAA). Exposure to 100 M AOAA totally inhibited GABA-T and all radioactivity released from slices was in authentic GABA. In studies on developing brain the 10-M concentration was also effective enough, except in cerebellar slices. In the absence of AOAA the major part of radioactivity spontaneously released from slices of adult cerebral cortex and cerebellum was tritiated water and still about one third part in the presence of 10 M AOAA. Potassium stimulation induced only the release of radioactive GABA but not labeled metabolites in both presence and absence of AOAA. AOAA reduced the stimulation-induced release of GABA. It is recommended that the use of GABA-T inhibitors should be discontinued in release experiments. Then labeled GABA must be separated in the effluents from its radioactive breakdown products.     

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.     

Gangliosides and regeneration of the goldfish optic nerve in vivo and in vitro.

One to forty days after optic nerve transection, goldfish received an i.p. injection of [3H]proline (proteins), 3HNAcGluc (gangliosides) or [3H]thymidine (DNA). After 1 or 2 days of incorporation, both optic systems were analyzed by biochemical and autoradiographical procedures. In the regenerating retina an enhanced retinal mitotic activity, protein synthesis (up to 2-fold) and ganglioside synthesis (up to 1.5-fold) was found. Simultaneously, a transiently enhanced accumulation (up to 4.5-fold) of axonally transported protein- and ganglioside-bound radioactivity in the regenerating optic nerve stump occurred. These regeneration-related proliferative and metabolic changes were found to be maximal at 6-8 days post lesion, but still measurable after 40 days. Concerning the endogenous ganglioside metabolism, in the regenerating retina no obvious change in ganglioside synthesis and composition could be observed, while in the regenerating optic nerve there was an enhanced accumulation of the ganglioside GP1c. Daily i.p. application of a ganglioside mixture from bovine brain (GMix) or of the monosialoganglioside GM1, did not alter significantly the degree and time course of the above regeneration induced metabolic changes or the regain of visual acuity. Sprouting activity of goldfish retinal explants was found to strongly depend upon a conditioning lesion of the optic nerve, reaching a maximum 8 days after nerve transection. This result strictly coincided with the profile of metabolic changes observed in vivo. Again, daily i.p. or i.o. injection of exogenous gangliosides did not influence the lesion induced increase of retinal sprouting activity. However, in normal, not regenerating animals, a local i.o. injection of GMix or GM1 led to a significant enhancement of the "basal" sprouting activity, normally occurring after lesion of the retina after injection of 0.9% NaCl. This ganglioside related stimulation was maximal at low concentrations (3 micrograms/eye) and did not occur at high concentrations (> 30 micrograms/eye). Injection of the phospholipid phosphatidylcholine or phosphatidylserine had no or a slightly inhibitory effect, when compared to NaCl controls. These data suggest an involvement of gangliosides in the complex process of induction of axonal sprouting.     

Abstracts: 1

Lead (Pb) is a known neurotoxic agent, however, mechanisms of its neurotoxicity are still an open question. The aim of the study was to assess the function of nerve endings and astroglia in regard to the relationships between GABA, glutamate and glutamine using the rodent model of Pb toxicity. Synaptosomal processes of radioactive neurotransmitter transport, both GABA and glutamate, were impaired. Moreover, the uptake of glutamine, involved in the neuronal recycling of the neurotransmitters GABA and glutamate, decreased. However, evidences of astroglial activation (enhanced uptake of glutamate and activation of glutamine synthetase) were observed, together with parallel existing weakness of glutamine transport. The results suggest that the disturbances in GABAergic and glutamateric neurotransmission may be partially the effect of impaired neuronal–astrocytic interaction, mainly in intercellular trafficking of glutamine.     

Alteration of zinc accumulation in ageing and neurodegenerative disorders

Lead (Pb) is a known neurotoxic agent, however, mechanisms of its neurotoxicity are still an open question. The aim of the study was to assess the function of nerve endings and astroglia in regard to the relationships between GABA, glutamate and glutamine using the rodent model of Pb toxicity. Synaptosomal processes of radioactive neurotransmitter transport, both GABA and glutamate, were impaired. Moreover, the uptake of glutamine, involved in the neuronal recycling of the neurotransmitters GABA and glutamate, decreased. However, evidences of astroglial activation (enhanced uptake of glutamate and activation of glutamine synthetase) were observed, together with parallel existing weakness of glutamine transport. The results suggest that the disturbances in GABAergic and glutamateric neurotransmission may be partially the effect of impaired neuronal–astrocytic interaction, mainly in intercellular trafficking of glutamine.