Glial Plasmalemmal Vesicles Research Articles

Neurochemical abnormalities in the hippocampus of male rats displaying audiogenic seizures, a genetic model of epilepsy

BackgroundEpilepsy is a disorder characterized by recurrent seizures that affects 1% of the population. However, the neurochemical alterations observed in epilepsy are not fully understood. There are different animal models of epilepsy, such as genetic or drug induced. In the present study, we utilize Wistar Audiogenic Rats (WAR), a murine strain that develops seizures in response to high intensity audio stimulation, in order to investigate abnormalities in glutamatergic and GABAergic systems. MethodsSynaptosomes and glial plasmalemmal vesicles were prepared from hippocampus and cortex, respectively. Glutamate and GABA release and uptake were assayed by monitoring the fluorescence and using L-[3H]-radiolabeled compounds. Glutamate and calcium concentration in the synaptosomes were also measured. The expression of neuronal calcium sensor 1 (NCS-1) was determined by western blot. ResultsGlutamate and GABA release evoked by KCl was decreased in WAR compared to control Wistar rats. Calcium independent release was not considerably different in both groups. The total amount of glutamate of synaptosomes, as well as glutamate uptake by synaptosomes and GPV were also decreased in WAR in comparison with the controls. In addition, [Ca2+]i of hippocampal synaptosomes, as well as NCS-1 expression in the hippocampus, were increased in WAR in comparison with controls. ConclusionIn conclusion, our results suggest that WAR have important alterations in the glutamatergic and GABAergic pathways, as well as an increased expression of NCS-1 in the hippocampus and inferior colliculus. These alterations may be linked to the spreading of hyperexcitability and recruitment of various brain regions.

Adenosine A2A receptors modulate glutamate uptake in cultured astrocytes and gliosomes

Glutamate is the primary excitatory neurotransmitter in the central nervous system, where its toxic build-up leads to synaptic dysfunction and excitotoxic cell death that underlies many neurodegenerative diseases. Therefore, efforts have been made to understand the regulation of glutamate transporters, which are responsible for the clearance of extracellular glutamate. We now report that adenosine A(2A) receptors (A(2A) R) control the uptake of D-aspartate in primary cultured astrocytes as well as in an ex vivo preparation enriched in glial plasmalemmal vesicles (gliosomes) from adult rats, whereas A(1) R and A(3) R were devoid of effects. Thus, the acute exposure to the A(2A) R agonist, CGS 21680, inhibited glutamate uptake, an effect prevented by the A(2A) R antagonist, SCH 58261, and abbrogated in cultured astrocytes from A(2A) R knockout mice. Furthermore, the prolonged activation of A(2A) R lead to a cAMP/protein kinase A-dependent reduction of GLT-I and GLAST mRNA and protein levels, which leads to a sustained decrease of glutamate uptake. This dual mechanism of inhibition of glutamate transporters by astrocytic A(2A) R provides a novel candidate mechanism to understand the ability of A(2) (A) R to control synaptic plasticity and neurodegeneration, two conditions tightly associated with the control of extracellular glutamate levels by glutamate transporters.

The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings.

The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.

Astrocytic response in the rodent model of global cerebral ischemia and during reperfusion

The present study investigated alterations in astrocytic cells after global cerebral ischemia resulting from cardiac arrest immediately and at several intervals after reperfusion when excessive formation of highly cytotoxic free radicals is known to occur. The cellular fraction of astrocytic origin (glial plasmalemmal vesicles - GPV) was examined by biochemical and immunochemical procedures. A tendency towards an elevation in immunocontent of glial fibrillary acidic protein (GFAP) was noticed after 24 hours whereas a significant increase was observed 7 days post ischemic event. The features of astrocytic stimulation were also observed in electron microscopy studies. An enhanced amount of gliofilaments was noticed in brain sections obtained from rats after 7 days of recovery. Simultaneously, a gradual decrease of total glutathione level, depending on the duration of reperfusion, was observed in brain homogenates and in fractions of astroglial origin. The most considerable reduction was observed on day 1 (52%) and day 7 (65%) after reperfusion in brain homogenates and on day 7 (47%) in GPV fraction. The results indicate an enhanced reactivity of astrocytic cells in ischemic conditions concomitantly with a long lasting decrease of total glutathione. Obviously, the inability of astrocytic glutathione system to detoxify free radicals formed during ischemic/reoxidation conditions may lead to damage to cerebral neurons by oxidative stress.

The protective role of astroglia in the early period of experimental lead toxicity in the rat

Although the clinical manifestations of lead (Pb) neurotoxicity are documented, the subcellular mechanisms of its action are still an open question. The purpose of this study was to assess the function of nerve ending particles after acute lead exposure and to investigate whether it exerts a toxic effect on astroglial functions. The studies were performed using the rodent model of acute lead toxicity. Cellular fractions were used in biochemical measurements - synaptosomes and glial plasmalemmal vesicles (GPV). Since a procedure for the isolation of the fraction of astroglial origin has been developed, it becomes possible to investigate lead-astroglia interactions after in vivo exposure. It is of importance because most of the studies concerning lead toxicity were performed using astroglial culture systems. It was found that the uptake of glutamate (Glu) to the synaptosomes was lowered and KCl-dependent release was increased, suggesting the impairment of glutamatergic transmission leading to the elevation of extracellular amino acid concentration. In contrast, glutamate uptake to the GPV fraction was significantly elevated. The activity of the marker enzyme - glutamine synthetase (GS) was also significantly increased in the GPV fraction. The activation of glial functions suggest a regulatory role for these cells in the early period of acute lead toxicity.

Reversible activation of glutamate transport in rat brain glia by protein kinase C and an okadaic acid-sensitive phosphoprotein phosphatase.

High-affinity L-glutamate (GLU) transport is an important regulator of excitatory amino acid (EAA) concentrations in brain extracellular fluid and may play a key role in excitatory synaptic transmission. In view of evidence that EAA transporters (EAAT) are heterogenous and contain consensus sites for phosphorylation, this investigation was undertaken to contrast the effects of transporter phosphorylation in fractions derived from glia and neurons (synaptosomes) of the adult rat forebrain. Treatment with phorbol-12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), increased the maximal rate of GLU transport in glial plasmalemmal vesicles by greater than 50 percent (237+/-18 vs. 365+/-27 pmol/mg protein/90s, p < 0.05) but caused no change in synaptosomes. The effect by PDBu was concentration and time-dependent and was inhibited completely by the PKC inhibitor calphostin C. Inhibition of serine-threonine phosphoprotein phosphatases with okadaic acid produced similar effects which were not additive with PDBu. Together, these results demonstrate that glial EAAT can be regulated by multiple phosphorylation processes.

Selective loss of phorbol-12,13-dibutyrate-facilitated L-glutamate transport in forebrain neurons of aged rats.

Phorbol-12,13-dibutyrate (PDBu), an activator of protein kinase C, was used to evaluate potential age-related changes in phosphorylation-dependent facilitation of high-affinity L-glutamate uptake in the rat central nervous system (CNS). Forebrain homogenates from male Fischer-344/brown Norway F1 hybrid rats were separated into glia-enriched (glial plasmalemmal vesicles) and neuron-enriched fractions (synaptosomes) and assayed for sodium-dependent transport of L-[3H]glutamate. Glial fractions from rats aged 5, 25, 31, and 37 months exhibited similar rates of basal L-[3H]glutamate transport and demonstrated no significant age-related differences with respect to the maximal facilitatory effect of PDBu (1-100 microM). In contrast, neuronal fractions exhibited an age-related decline in both indices, with basal L-[3H]glutamate transport decreasing from 710+/-31 to 560+/-40 pmoL/mg protein/90 s for the 5- and 37-month groups, respectively (p < .03) and PDBu having a significantly attenuated effect in aged animals. Together, these results provide support for the hypothesis that aging is associated with a decrease in the number of neuronal L-glutamate transporters as well as a diminished capacity to up-regulate these transporters through a protein kinase C-dependent pathway.

Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles

Cultured astrocytes derived from neonatal rat brain exhibited high affinity, Na+-dependent, paroxetine and fluoxetine sensitive [3H] 5-HT uptake. Reverse transcriptase-PCR demonstrated that astrocytes in culture expressed messenger RNA for the cloned serotonin transporter protein which has been characterised as the neuronal serotonin transporter. Although the serotonin transporter in cultured astrocytes displayed a Km value approximately 10 times greater than found in adult brain synaptosomes, these observations indicated that astrocytes in vitro may express the same serotonin transporter as neurons. Reverse transcriptase-PCR demonstrated the presence of serotonin transporter mRNA in the adult rat cerebral cortex, suggesting that astrocytes in vivo may express low levels of this mRNA. To investigate whether astrocytes in the adult CNS express functional serotonin transporters, glial plasmalemmal vesicles were prepared from cerebral cortex, representing a subcellular fraction composed primarily of vesicles derived from astrocytes. These vesicles were characterised by [3H]-glutamate and [3H]-dopamine uptake and by immunoblot analysis, using glial and synaptic markers: glutamate synthase, SNAP-25 and synaptobrevin. [3H]5-HT was taken up into glial plasmalemmal vesicles in a high affinity (Km approximately 40 nM), Na + dependent, paroxetine-sensitive manner. The [3H]5-HT uptake capacity (Vmax ) in these vesicles was approximately one quarter of that observed in synaptosomes. These data indicate that astrocytes in culture and in vivo are capable of 5-HT uptake via the previously characterised ‘neuronal’ serotonin transporter.

Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles

Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles

Increase of glutamate uptake in astrocytes: a possible mechanism of action of volatile anesthetics.

Glutamate is the most ubiquitous excitatory neurotransmitter in the vertebrate central nervous system. Astrocytes play an important role in terminating glutamatergic neurotransmission by removing released glutamate from the synaptic cleft. The authors examined the effects of several anesthetics on the glutamate uptake activity of astrocytes. Cultured astrocytes from hippocampi of rat embryos were incubated with solution containing [3H]glutamate, which was pre-equilibrated with 0-4% halothane at 37 degrees C. The uptake activity was evaluated as the amount of radioactivity per cell of protein. When the reaction solution was equilibrated with 4% halothane, glutamate uptake increased to about 165% of the control. The effect of halothane was dose-dependent, and a significant augmentation (30-50%) of glutamate uptake was observed at a range in clinical use concentrations (1-2%). On the other hand, the uptake of gamma-aminobutyric acid, an inhibitory transmitter, was hardly affected by 1-4% halothane. The effect of halothane on glutamate uptake was also examined in neuron-rich culture, and similar augmentation was observed, although the extent was less than that in astrocyte culture. Biochemical subcellular fractions (i.e., glial plasmalemmal vesicles and synaptosomes) were also examined, however, only slight (not significant) increase was detected in the glutamate uptake activity. Other volatile anesthetics, such as enflurane, isoflurane, and sevoflurane, also enhanced glutamate uptake, whereas the intravenous anesthetics ketamine and pentobarbital showed no effect on glutamate uptake. The increase of glutamate uptake by astrocytes in the presence of volatile anesthetics potentially attenuates excitatory synaptic transmission in the entire central nervous system, a finding that may explain in part the action of volatile anesthetics.

Transport mechanism of glutamate by hypotonic-treated glial plasmalemmal vesicles from rat hippocampus. Effects of concentration gradients of Na+ and K+ and of ionophores.

Recently, we isolated a novel subcellular fraction of glial plasmalemmal vesicles (GPV), which showed a higher activity of Na(+)-dependent glutamate transport than synaptosomes (Nakamura et al., 1993). In order to study kinetically the glutamate transport mechanism, we measured the reaction under various ionic conditions both inside and outside the vesicles. The vesicles treated hypotonically and preloaded with KCl could take up glutamate in the presence of external Na+. The level of glutamate uptake was dependent on external concentrations of NaCl ([NaCl]o) and competitively inhibited by [KCl]o. However, it was dependent on [KCl]i, and competitively inhibited by [NaCl]i. The activation and inhibition constants of K+ were about 30 mM inside and 20 mM outside, respectively, whereas those of Na+ were 140 mM outside and 4 mM inside, respectively. These results suggest that the transport carrier molecules work asymmetrically to the membranes. Nigericin and monensin, acidic ionophores for K+ and Na+, respectively, inhibited the glutamate uptake. On the other hand, valinomycin, a neutral ionophore for K+, elevated the uptake level, suggesting that the inside-negative membrane potential induced by K+ diffusion enhances the uptake activity. We conclude that glutamate transport by glial cells requires both external Na+ and internal K+ and is regulated by the membrane potential.

(2 S,3 S,4 R)-2-(Carboxycyclopropyl)glycine, a potent and competitive inhibitor of both glial and neuronal uptake of glutamate

The effects of several diastereoisomers of L-2-(carboxycyclopropyl)glycine (CCG) on L-glutamate uptake were compared among three different preparations, glial plasmalemmal vesicles (GPV), synaptosomes and cultured astrocytes from rat hippocampus. The (2 S,3 S,4 R)-isomer ( L-CCG-III) inhibited a Na +-dependent high-affinity L-glutamate uptake in GPV and synaptosomes in a dose dependent manner at a micromolar range. The potency was quite similar to that of L- threo-β-hydroxyaspartate in both subcellular fractions and much higher than L-aspartate-β-hydroxamate, which were known as potent inhibitors of glutamate uptake. The (2 S,3 S,4 R)-isomer ( L-CCG-IV) also inhibited the glutamate uptake in GPV and synaptosomes, but it was about 100 times less active than L-CCG-III. The (2 S,3 S,4 R)- and (2 S,3 R,4 R)-isomers ( L-CCG-I and L-CCG-II, respectively) hardly showed any inhibitory action on the glutamate uptake. Dixon plot analysis of the initial uptake rate revealed that the inhibition was in a competitive manner and the value of the inhibition constant ( K i ) was about 1 μM in both GPV and synaptosomes. L-CCG-III effectively inhibited the glutamate uptake by cultured hippocampal astrocytes as well. These results suggested that L-CCG-III inhibited the glutamate uptake in both neurones and glial cells of the mammalian central nervous system in a similar manner.

Glial plasmalemmal vesicles: A subcellular fraction from rat hippocampal homogenate distinct from synaptosomes

By a Percoll density-gradient centrifugation of rat hippocampal homogenate, we found a novel subcellular fraction (specific gravity approximately 1.046 g/ml), besides synaptosomes (approximately 1.060 g/ml), which showed a high activity of Na(+)-dependent glutamate uptake. The initial rate of the glutamate uptake in this fraction was as high as twice that in synaptosomes. Activities of choline acetyltransferase and high affinity choline uptake were, on the other hand, much lower. gamma-Aminobutyric acid uptake activity was nearly equivalent in both fractions. Electron microscopic observations revealed that the fraction was morphologically different from synaptosomal or myelin fractions, but mainly consisted of two different types of empty membrane vesicles; irregular (0.3-0.8 micron in diameter) and spheroid type (0.2 micron). The immunoreactivity to glial fibrillary acidic protein was appreciably high in this fraction. The marker enzyme analysis showed the fraction was rich in plasma membranes. On the basis of these results, the fraction is termed glial plasmalemmal vesicles (GPV). We analyzed kinetically the reaction of Na(+)-dependent glutamate uptake by GPV comparing with that by synaptosomes. Km values for glutamate in GPV was 4.7 microM and Vmax was 33 nmol/mg/min, while in synaptosomes 11 microM and 17 nmol/mg/min, respectively. Hill coefficients of Na+ activation in GPV and synaptosomes were 1.1 and 2.0, respectively. Thus, the mechanism or transporter molecule in glial cells for Na(+)-dependent glutamate transport is likely to be different from that in neurons.