Rat Synaptosomes Research Articles

Effects of antiepileptic drugs on GABA release from rat and human neocortical synaptosomes

In epilepsy, allegedly, a neurotransmitter imbalance between the inhibitory GABA and the excitatory glutamate prevails. Therefore, some antiepileptic drugs (AEDs) are thought to increase GABA release. Because little is known about corresponding presynaptic effects of AEDs in the human brain, this study investigated the effects of carbamazepine, lamotrigine, phenytoin, gabapentin, pregabalin, levetiracetam, and valproate on (3)H-GABA release from human neocortical synaptosomes preincubated with (3)H-GABA. To obtain information on possible species differences, rat neocortical synaptosomes were investigated concomitantly. Release was evoked by either veratridine (1, 3.2, or 10 μM), which prevents activated voltage-dependent Na(+) channels from closing, or elevation of extracellular [K(+)] from 3 to 15 mM. The exocytosis inhibitor tetanus toxin (TeT) or withdrawal of buffer Ca(2+) (Ca (e) (2+) ) reduced K(+)-evoked release in both species, while blockade of Na(+) channels with tetrodotoxin had no effect. K(+)-evoked release was characterized as predominant, Ca(2+)-dependent and Na(+)-independent, exocytosis. Carbamazepine and phenytoin in the rat and carbamazepine, phenytoin, lamotrigine, and valproate in human tissue reduced K(+)-evoked (3)H-GABA release. With respect to veratridine-evoked release, Ca (e) (2+) withdrawal did not reduce release in the rat; it even increased the release in human tissue. TeT was slightly inhibitory in the rat. Blockade of GABA transport diminished veratridine-evoked (3)H-GABA release in either species. This release was characterized as mediated mainly by transporter reversal. Carbamazepine, lamotrigine, and phenytoin in rat tissue and carbamazepine and phenytoin in human decreased veratridine-induced (3)H-GABA release. Interestingly, no AED increased (3)H-GABA release. The reduction by AEDs of veratridine-evoked release was more intense than that of K(+)-evoked release. In conclusion, reduction of GABA release by AEDs may be the actual objective in a pathologically altered neuronal network where GABA acts in a depolarizing fashion.

Effects of antiepileptic drugs on glutamate release from rat and human neocortical synaptosomes

Aim of this study was to learn whether the antiepileptic drugs (AEDs) carbamazepine, lamotrigine, phenytoin, gabapentin, pregabalin, levetiracetam, and valproate (all at 100 μM) presynaptically influence the neurotransmission of the excitatory transmitter glutamate. The effects of these frequently used AEDs were examined on ³H-glutamate release from superfused synaptosomes of both rat and human neocortex. Release was evoked by elevation of buffer [K(+)] from 3 to 15 mM or by the Na(+) channel activator veratridine (1, 3.2, and 10 μM). Buffer [K(+)] elevation induced ³H-glutamate exocytosis, which was Ca(²+)-, but not Na(+)-, dependent and which was accompanied only in human tissue by release through transporter reversal. In rat tissue, release was diminished by the Na(+) channel inhibitors carbamazepine, lamotrigine, and phenytoin, which therefore may also affect presynaptic Ca(²+) channels. Interestingly, levetiracetam increased ³H-glutamate release. In contrast, the tested AEDs did not affect K(+)-evoked ³H-glutamate release in human tissue, neither when the transporters were operative nor when exocytosis was isolated by transporter blockade. Veratridine-evoked ³H-glutamate release was a Na(+)-dependent transmitter efflux through reversed transporters in both species which in human synaptosomes was accompanied by exocytosis. The latter depended on external Ca(²+). Carbamazepine, lamotrigine, and phenytoin reduced this release from both rat and human tissue. There is an obvious species difference in the effects of carbamazepine, lamotrigine, and phenytoin on K(+)-evoked ³H-glutamate release while their inhibitory effects on veratridine-evoked release were similar. Thus, the depression of ³H-glutamate release by carbamazepine, lamotrigine, and phenytoin may be due to inhibited synaptosomal Na(+) or Ca(²+) influx.

P2Y Purinergic Regulation of the Glycine Neurotransmitter Transporters

The sodium- and chloride-coupled glycine neurotransmitter transporters (GLYTs) control the availability of glycine at glycine-mediated synapses. The mainly glial GLYT1 is the key regulator of the glycine levels in glycinergic and glutamatergic pathways, whereas the neuronal GLYT2 is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft. In this study, we report that stimulation of P2Y purinergic receptors with 2-methylthioadenosine 5'-diphosphate in rat brainstem/spinal cord primary neuronal cultures and adult rat synaptosomes leads to the inhibition of GLYT2 and the stimulation of GLYT1 by a paracrine regulation. These effects are mainly mediated by the ADP-preferring subtypes P2Y(1) and P2Y(13) because the effects are partially reversed by the specific antagonists N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate and pyridoxal-5'-phosphate-6-azo(2-chloro-5-nitrophenyl)-2,4-disulfonate and are totally blocked by suramin. P2Y(12) receptor is additionally involved in GLYT1 stimulation. Using pharmacological approaches and siRNA-mediated protein knockdown methodology, we elucidate the molecular mechanisms of GLYT regulation. Modulation takes place through a signaling cascade involving phospholipase C activation, inositol 1,4,5-trisphosphate production, intracellular Ca(2+) mobilization, protein kinase C stimulation, nitric oxide formation, cyclic guanosine monophosphate production, and protein kinase G-I (PKG-I) activation. GLYT1 and GLYT2 are differentially sensitive to NO/cGMP/PKG-I both in brain-derived preparations and in heterologous systems expressing the recombinant transporters and P2Y(1) receptor. Sensitivity to 2-methylthioadenosine 5'-diphosphate by GLYT1 and GLYT2 was abolished by small interfering RNA (siRNA)-mediated knockdown of nitric-oxide synthase. Our data may help define the role of GLYTs in nociception and pain sensitization.

Acetate-Dependent Mechanisms of Inborn Tolerance to Ethanol

To clarify the role of acetate in neurochemical mechanisms of the initial (inborn) tolerance to ethanol. Rats with low and high inborn tolerance to hypnotic effect of ethanol were used. In the brain region homogenates (frontal and parietal cortex, hypothalamus, striatum, medulla oblongata) and brain cortex synaptosomes, the levels of acetate, acetyl-CoA, acetylcholine (AcH), the activity of pyruvate dehydrogenase (PDG) and acetyl-CoA synthetase were examined. It has been found that brain cortex of rats with high tolerance to hypnotic effect of ethanol have higher level of acetate and activity of acetyl-CoA synthetase, but lower level of acetyl-СCoA and activity of PDG. In brain cortex synaptosomes of tolerant rats, the pyruvate oxidation rate as well as the content of acetyl-CoA and AcH synthesis were lower when compared with intolerant animals. The addition of acetate into the medium significantly increased the AcH synthesis in synaptosomes of tolerant, but not of intolerant animals. Calcium ions stimulated the AcH release from synaptosomes twice as high in tolerant as in intolerant animals. Acetate eliminated the stimulating effect of calcium ions upon the release of AcH in synaptosomes of intolerant rats, but not in tolerant animals. As a result, the quantum release of AcH from synaptosomes in the presence of acetate was 6.5 times higher in tolerant when compared with intolerant rats. The brain cortex of rats with high inborn tolerance to hypnotic effect of ethanol can better utilize acetate for the acetyl-CoA and AcH synthesis, as well as being resistant to inhibitory effect of acetate to calcium-stimulated release of AcH. It indicates the metabolic and cholinergic mechanisms of the initial tolerance to ethanol.

Characterization of Pharmacological and Wake-Promoting Properties of the Dopaminergic Stimulant Sydnocarb in Rats

Sydnocarb is a psychomotor stimulant structurally similar to d-amphetamine (D-AMPH) and is used in Russia for the treatment of a variety of neuropsychiatric comorbidities. The nature of sydnocarb-induced facilitation of dopamine (DA) neurotransmission [DA release versus DA transporter (DAT) inhibition] is not clear. The present study characterized the pharmacological actions and behavioral effects of intraperitoneal sydnocarb in male Sprague-Dawley rats. Where relevant, comparisons were made with intraperitoneal D-AMPH. Unlike D-AMPH, which causes release of DA from rat synaptosomes (EC(50) = 0.10 μM; 95% confidence limits, 0.06-0.18), sydnocarb (up to 100 μM) did not. Sydnocarb potently (K(i) = 8.3 ± 0.7 nM) blocked recombinant human DAT expressed in Chinese hamster ovary-K1 cells and less potently blocked the norepinephrine transporter (K(i) = 10.1 ± 1.5 μM). Sydnocarb at 10 μM did not bind to 64 other targets. In rats, 10 and 30 mg/kg sydnocarb showed a 2-fold longer half-life in plasma and brain and a 5-fold lower brain-to-plasma ratio compared with 0.3 and 1 mg/kg D-AMPH. In the Irwin assay, sydnocarb was well tolerated up to 30 mg/kg; D-AMPH-like stereotypic behaviors were evident at 100 mg/kg. Behavioral effects of 30 mg/kg sydnocarb and 0.3 mg/kg D-AMPH were comparable. In a sleep/wake assay, 10 mg/kg sydnocarb and 1 mg/kg D-AMPH increased wakefulness comparably; however, sydnocarb (up to 30 mg/kg) did not induce D-AMPH-like rebound hypersomnolence (RHS). Like D-AMPH, sydnocarb enhanced theta power, an electrophysiological measure of cognitive function. In conclusion, sydnocarb is a selective and potent DAT inhibitor that produces robust increases in the wake state without RHS, and with potential cognitive-enhancing properties.

RP-LC with Fluorescence Detection of Amino Acids in Rat Brain Synaptosomes

For the first time, a simple and validated reversed-phase liquid chromatography (RP-LC) with fluorescence detection has been developed for the simultaneous analysis of glutamate (Glu), γ-aminobutyric acid (GABA), glycine (Gly) and taurine (Tau) in Wistar and tremor rats brain synaptosomes. The samples were separated on a C18 analytical column with gradient elution of methanol and 0.1 mol L−1 potassium acetate at a flow rate of 1 mL min−1. Total run time was approximately 25 min. All calibration curves exhibited good linearity (r 2 > 0.999) within test ranges. The reproducibility was estimated by intra-and inter-day assays and RSD values were less than 2.48%. The recoveries were between 96.32 and 105.21%. The method was successfully applied to the quantification of amino acids in Wistar and tremor rats brain synaptosomes. Through this developed protocol, the levels of Glu in hippocampal and prefrontal cortical synaptosomes of tremor rats were both significantly elevated than those of adult Wistar rats whereas significantly decreased concentrations of GABA and Gly were observed in the hippocampal region of tremor rats without evident difference in the prefrontal cortex between experimental and control groups. In addition, our studies also showed a marked elevation of Tau in tremor rats hippocampal synaptosomes although there was no pronounced difference in the prefrontal cortical region of Wistar and tremor rats.

Insulin modifies aging-related inhibition of 1-stearoyl, 2-arachidonoylglycerol phosphorylation in rat synaptic terminals

The purpose of the present study was to analyze diacylglycerol kinase (DAGK) activity in synaptic terminals from cerebral cortex (CC) and hippocampus (Hp) from adult (3–4 month-old) and aged (26–28 month-old) rats. The effect of insulin through DAGK activity on synaptosomes from adult and aged rats was also analyzed under conditions favoring saturated or unsaturated phosphatidic acid (PA) formation, using exogenous di-palmitoil glycerol (DPG) or 1-stearoyl-2-arachidonoylglycerol (SAG) as substrates. Results showed that the enzymatic activity preferentially uses SAG as substrate, thus indicating the presence of ɛ-type DAGK. A significant decrease in DAGK activity transforming SAG into PA was also observed in both tissues from aged rats. Western blot detection of DAGKɛ showed that enzyme content undergoes no changes with aging.[3H] inositol incorporation into phosphoinosites was also analyzed to evaluate the role of DAGKɛ in their synthesis. Data obtained from 3H-inositol incorporation into phosphoinositides revealed that in synaptosomes from aged rats phosphatidylinositol (PI) synthesis is lower than in adult animals. Interestingly, in the presence of SAG, PI synthesis was restored to adult values.DAGK activity over SAG was more highly stimulated by insulin in CC and Hp synaptosomes of aged rats with respect to adult rats. On the other hand, insulin exerted a stimulatory effect on PI and phosphatidylinositol 4 phosphate (PI(4)P) synthesis in synaptosomal CC from aged rats. Taken together, our findings indicate that in aged rats insulin triggers a stimulatory mechanism that reverts the diminished synaptosomal ability to synthesize arachidonoyl phosphatidic acid (20:4 PA). The recovery of this PA species indicates that insulin positively regulates phosphoinositide synthesis.

Pre‐synaptic BK channels selectively control glutamate versus GABA release from cortical and hippocampal nerve terminals

In the present study, by means of genetic, biochemical, morphological, and electrophysiological approaches, the role of large-conductance voltage- and Ca(2+)-dependent K(+) channels (BK channels) in the release of excitatory and non-excitatory neurotransmitters at hippocampal and non-hippocampal sites has been investigated. The results obtained show that the pharmacological modulation of pre-synaptic BK channels selectively regulates [(3)H]D-aspartate release from cortical and hippocampal rat synaptosomes, but it fails to influence the release of excitatory neurotransmitters from cerebellar nerve endings or that of [(3)H]GABA, [(3)H]Noradrenaline, or [(3)H]Dopamine from any of the brain regions investigated. Confocal immunofluorescence experiments in hippocampal or cerebrocortical nerve terminals revealed that the main pore-forming BK α subunit was more abundantly expressed in glutamatergic (vGLUT1(+)) versus GABAergic (GAD(65-67)(+)) nerve terminals. Double patch recordings in monosynaptically connected hippocampal neurons in culture confirmed a preferential control exerted by BK channels on glutamate over GABA release. Altogether, the present results highlight a high degree of specificity in the regulation of the release of various neurotransmitters from distinct brain regions by BK channels, supporting the concept that BK channel modulators can be used to selectively limit excessive excitatory amino acid release, a major pathogenetic mechanism in several neuropsychiatric disorders.

Differential inhibitory effects of drugs acting at the noradrenaline and 5‐hydroxytryptamine transporters in rat and human neocortical synaptosomes*

Although the amino acid sequences of rat and human 5-hydroxytryptamine (5-HT) and noradrenaline (NA) transporters (i.e. SERT and NET) are highly homologous, species differences exist in the inhibitory effects of drugs acting at these transporters. Therefore, comparison of the potencies of drugs acting at SERT and NET in native human and rat neocortex may serve to more accurately predict their clinical profile. Synaptosomes prepared from fresh human and rat neocortical tissues were used for [(3)H]-5-HT and [(3)H]-NA saturation and competition uptake experiments. The drugs tested included NA reuptake inhibitors (desipramine, atomoxetine and (S,S)-reboxetine), 5-HT reuptake blockers (citalopram, fluoxetine and fluvoxamine) and dual 5-HT/NA reuptake inhibitors (duloxetine and milnacipran). In saturation experiments on synaptosomal [(3)H]-5-HT and [(3)H]-NA uptake, the dissociation constants did not indicate species differences although a smaller density of both SERT and NET was observed in human tissues. In competition experiments with the various drugs, marked species differences in their potencies were observed, especially at SERT. The rank order of selectivity ratios (SERT/NET) in human neocortex was as follows: citalopram >or= duloxetine = fluvoxamine >or= fluoxetine > milnacipran > desipramine = atomoxetine > (S,S)-reboxetine. Significant species differences in these ratios were observed for duloxetine, atomoxetine and desipramine. This study provides the first compilation of drug potency at native human neocortical SERT and NET. The significant species differences (viz., human vs. rat) in drug potency suggest that the general use of rodent data should be limited to predict clinical efficacy or profile.

The Roles of Dopamine Transport Inhibition and Dopamine Release Facilitation in Wake Enhancement and Rebound Hypersomnolence Induced by Dopaminergic Agents

Rebound hypersomnolence (RHS: increased sleep following increased wake) is a limiting side-effect of many wake-promoting agents. In particular, RHS in the first few hours following wake appears to be associated with dopamine (DA)-releasing agents, e.g., amphetamine, but whether it can also be produced by DA transporter (DAT) inhibition alone is unknown. In these studies, DA-releasing and DAT-inhibiting agents and their interaction were systematically examined for their ability to increase wake and induce RHS. Chronically implanted rats were evaluated in a blinded, pseudo-randomized design. 237 rats were used in these studies with 1 week between repeat tests. Animals were habituated overnight and dosed the next day, 5 h after lights on, with test agents. Sleep/wake activityand RHS were evaluated using EEG/EMG recording up to 22 h post dosing. In vitro dopamine release was evaluated in rat synaptosomes. At doses that produced equal increases in wake, DA-releasing (amphetamine, methamphetamine, phentermine) and several DAT-inhibiting agents (cocaine, bupropion, and methylphenidate) produced RHS during the first few hours after the onset of sleep recovery. However, other DAT-inhibiting agents (mazindol, nomifensine, GBR-12909, and GBR-12935) did not produce RHS. Combination treatment with amphetamine and nomifensine produced waking activity greater than the sum of their individual activities alone while ameliorating the amphetamine-like RHS. In rat synaptosomes, nomifensine reduced the potency of amphetamine to induce DA release approximately 270-fold, potentially explaining its action in ameliorating amphetamine-induced RHS. All DA releasing agents tested, and some DAT-inhibiting agents, produced RHS at equal wake-promoting doses. Thus amphetamine-like DA release appears sufficient for inducing RHS, but additional properties (pharmacologic and/or pharmacokinetic) evidently underlie RHS of other DAT inhibitors. Enhancing wake while mitigating RHS can be achieved by combining DAT-inhibiting and DA-releasing agents.

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Sevoflurane and propofol depolarize mitochondria in rat and human cerebrocortical synaptosomes by different mechanisms

The mitochondrial membrane potential drives the main functions of the mitochondria. Sevoflurane depolarizes neural mitochondria. There is still, however, limited information concerning the effect of anaesthetics on neural mitochondria in humans. The effect of sevoflurane and propofol on the intracellular Ca(2+) concentration [Ca(2+)](i) and the mitochondrial membrane potential (DeltaPsi(m)) was therefore compared in rat and human synaptosomes, and the changes were related to interventions in the electron transport chain. Synaptosomes from rat and human cerebral cortex were loaded with the fluorescent probes fura-2 ([Ca(2+)](i)) and JC-1 (DeltaPsi(m)) before exposure to sevoflurane 1 and 2 minimum alveolar concentration (MAC), and propofol 30 and 100 microM. The effect on the electron transport chain was investigated by blocking complex V. Sevoflurane and propofol decreased DeltaPsi(m) in rat synaptosomes in a dose-dependent manner, and to the same extent by equipotent doses. Inhibition of complex V enhanced the depolarizing effect of sevoflurane 2 MAC, but not of propofol 100 microM. Neither sevoflurane nor propofol affected [Ca(2+)](i) significantly. Sevoflurane and propofol decreased DeltaPsi(m) in human synaptosomes to the same extent as in the rat experiments. Sevoflurane and propofol at equipotent doses depolarize the mitochondria in rat and human nerve terminals to the same extent. The depolarizing effect of propofol on Psi(m) was more rapid in onset than that of sevoflurane. Whereas sevoflurane inhibits the respiratory chain sufficiently to cause ATP synthase reversal, the depolarizing effect of propofol seems to be related to inhibition of the respiratory chain from complex I to V.

Ectonucleotidase and acetylcholinesterase activities in synaptosomes from the cerebral cortex of streptozotocin-induced diabetic rats and treated with resveratrol

The aim of the present study was to investigate the effects of resveratrol (RV), an important neuroprotective compound on NTPDase, 5′-nucleotidase and acetylcholinesterase (AChE) activities in cerebral cortex synaptosomes of streptozotocin (STZ)-induced diabetic rats. The animals were divided into six groups ( n = 8): control/saline; control/RV 10 mg/kg; control/RV 20 mg/kg; diabetic/saline; diabetic/RV 10 mg/kg; diabetic/RV 20 mg/kg. After 30 days of treatment with resveratrol the animals were sacrificed and the cerebral cortex was removed for synaptosomes preparation and enzymatic assays. The results demonstrated that NTPDase and 5′-nucleotidase activities were significantly increased in the diabetic/saline group ( p < 0.05) compared to control/saline group. Treatment with resveratrol significantly increased NTPDase, 5′-nucleotidase activities in the diabetic/RV10 and diabetic/RV20 groups ( p < 0.05) compared to diabetic/saline group. When resveratrol was administered per se there was also an increase in the activities of these enzymes in the control/RV10 and control/RV20 groups ( p < 0.05) compared to control/saline group. AChE activity was significantly increased in the diabetic/saline group ( p < 0.05) compared to control/saline group. The treatment with resveratrol prevented this increase in the diabetic/RV10 and diabetic/RV20 groups. In conclusion, this study demonstrated that the resveratrol interfere with the purinergic and cholinergic neurotransmission by altering NTPDase, 5′-nucleotidase and AChE activities in cerebral cortex synaptosomes of diabetic rats. In this context, we can suggest that resveratrol should be considered potential therapeutics and scientific tools to be investigated in brain disorders associated with the diabetes.

Synaptopathy under conditions of altered gravity: Changes in synaptic vesicle fusion and glutamate release

Glutamate release and synaptic vesicle heterotypic/homotypic fusion were characterized in brain synaptosomes of rats exposed to hypergravity (10 G, 1 h). Stimulated vesicular exocytosis determined as KCl-evoked fluorescence spike of pH-sensitive dye acridine orange (AO) was decreased twice in synaptosomes under hypergravity conditions as compared to control. Sets of measurements demonstrated reduced ability of synaptic vesicles to accumulate AO (∼10% higher steady-state baseline level of AO fluorescence). Experiments with preloaded l-[ 14C]glutamate exhibited similar amount of total glutamate accumulated by synaptosomes, equal concentration of ambient glutamate, but the enlarged level of cytoplasmic glutamate measuring as leakage from digitonin-permeabilized synaptosomes in hypergravity. Thus, it may be suggested that +G-induced changes in stimulated vesicular exocytosis were a result of the redistribution of intracellular pool of glutamate, i.e. a decrease in glutamate content of synaptic vesicles and an enrichment of the cytoplasmic glutamate level. To investigate the effect of hypergravity on the last step of exocytosis, i.e. membrane fusion, a cell-free system consisted of synaptic vesicles, plasma membrane vesicles, cytosolic proteins isolated from rat brain synaptosomes was used. It was found that hypergravity reduced the fusion competence of synaptic vesicles and plasma membrane vesicles, whereas synaptosomal cytosolic proteins became more active to promote membrane fusion. The total rate of homo- and heterotypic fusion reaction initiated by Ca 2+ or Mg 2+/ATP remained unchanged under hypergravity conditions. Thus, hypergravity could induce synaptopathy that was associated with incomplete filling of synaptic vesicles with the neuromediator and changes in exocytotic release.

Effect of thyroid hormone T3 on Myosin-Va expression in the central nervous system

Thyroid hormones (THs) are essential for brain development, where they regulate gliogenesis, myelination, cell proliferation and protein synthesis. Hypothyroidism severely affects neuronal growth and establishment of synaptic connections. Triiodothyronine (T3), the biologically active form of TH, has a central function in these activities. So, Myosin-Va (Myo-Va), a molecular motor protein involved in vesicle and RNA transport, is a good candidate as a target for T3 regulation. Here, we analyzed Myo-Va expression in euthyroid and hypothyroid adult rat brains and synaptosomes. We observed a reduction of Myo-Va expression in cultured neural cells from newborn hypothyroid rat brain, while immunocytochemical experiments showed a punctate distribution of this protein in the cytoplasm of cells. Particularly, Myo-Va co-localized with microtubules in neurites, especially in their varicosities. Myo-Va immunostaining was stronger in astrocytes and neurons of controls when compared with hypothyroid brains. In addition, supplementation of astrocyte cultures with T3 led to increased expression of Myo-Va in cells from both euthyroid and hypothyroid animals, suggesting that T3 modulates Myo-Va expression in neural cells both in vivo and in vitro. We have further analyzed Myo-Va expression in U373 cells, a human glioblastoma line, and found the same punctate cytoplasmic protein localization. As in normal neural cells, this expression was also increased by T3, suggesting that the modulatory mechanism exerted by T3 over Myo-Va remains active on astrocyte tumor cells. These data, coupled with the observation that Myo-Va is severely affected in hypothyroidism, support the hypothesis that T3 activity regulates neural motor protein expression, taking Myo-Va as a model. As a consequence, reduced T3 activity could supposedly affect axonal transport and synaptic function, and could therefore explain disturbances seen in the hypothyroid brain.

Inhibition of Ca2+‐dependent glutamate release from cerebral cortex synaptosomes of rats with experimental autoimmune encephalomyelitis

Several pathological studies have revealed a prominent involvement of the cerebral cortex in patients with multiple sclerosis (MS). In order to better understand the events that lead to the progressive neuronal dysfunction in MS, herein we explore the contribution of the glutamatergic release in cerebral cortex synaptosomes isolated from rats with experimental autoimmune encephalomyelitis, an animal model reproducing many features of MS. We found that the Ca(2+)-dependent but not the Ca(2+)-independent glutamate release induced by KCl and 4-aminopyridine was significantly decreased during the acute stage of the disease. This inhibited release coincides with the onset of the clinical signs and after 24 h tends to recover the level of the control animals. The results also showed an inhibition of the glutamate release stimulated by ionomycin. When the animals were totally recovered from clinical signs, the neurotransmitter release stimulated by the different inductors was similar to the controls. Examination of the cytosolic Ca(2+) using fura-2-acetoxymethyl ester revealed that the inhibition of glutamate release could not be attributed to a reduction in voltage-dependent Ca(2+) influx. However, this inhibition was concomitant with a lower phosphorylation of synapsin I at P-site1. Our results show that the inhibition observed on the Ca(2+)-dependent neurotransmitter release from cerebral cortex synaptosomes in experimental autoimmune encephalomyelitis is specific and correlates with the beginning of the clinical disease. Moreover, they suggest an alteration in the metabolism of proteins involved in the vesicular glutamate release more than a deregulation in the influx of cytosolic Ca(2+).

Disubstituted Diaryl Diselenides Inhibit [3H]-Serotonin Uptake in Rats

Diphenyl diselenide (PhSe)2, the parent compound of disubstituted diaryl diselenides, produces significant antidepressant-like effect when evaluated in the forced swimming test (FST) in rats. The goal of this study was to investigate the effects of diphenyl diselenide (PhSe)2, m-trifluoromethyl-diphenyl diselenide (F3CPhSe)2 and p-methoxy-diphenyl diselenide (CH3OPhSe)2 on [3H]-serotonin uptake in rat platelets and synaptosomes. At concentrations higher than 10 microM (PhSe)2, and (F3CPhSe)2 inhibited about 20% [3H]-serotonin uptake in platelets. (CH3OPhSe)2 inhibited [3H]-serotonin uptake in platelets at concentrations higher than 1 microM. Similarly, (PhSe)2, (F3CPhSe)2, and (CH3OPhSe)2 significantly inhibited [3H]-serotonin uptake (about 50%) in synaptosomes from rat brain at concentrations higher than 1 microM. The maximal inhibitory effects of (PhSe)2, (F3CPhSe)2, and (CH3OPhSe)2 on [3H]-serotonin uptake from rat synaptosomes were 65%, 96%, and 80%, respectively. In conclusion, our results demonstrate that disubstituted diaryl diselenides altered monoaminergic system by interacting with monoamine uptake.

NMDA‐mediated modulation of dopamine release is modified in rat prefrontal cortex and nucleus accumbens after chronic nicotine treatment

In this study, we investigate the effects of chronic administration of (-)nicotine on the function of the NMDA-mediated modulation of [(3)H]dopamine (DA) release in rat prefrontal cortex (PFC) and nucleus accumbens (NAc). In the PFC synaptosomes NMDA in a concentration-dependent manner evoked [(3)H]DA release in rats chronically treated with vehicle (14 days) with an EC(50) of 13.1 +/- 2.0 microM. The NMDA-evoked overflow of the [(3)H]DA in PFC nerve endings of rats treated with (-)nicotine was significantly lower (-43%) than in vehicle treated rats. The EC(50) was 9.0 +/- 1.4 microM. Exposure of NAc synaptosomes of rats treated with vehicle to NMDA produced an increase in [(3)H]DA overflow with an EC(50) of 14.5 +/- 5.5 microM. This effect was significantly enhanced in chronically treated animals. The EC(50) was 10.5 +/- 0.5 microM. The K(+)-evoked release of [(3)H]DA was not modified by the (-)nicotine administration. Both the changes of the NMDA-evoked [(3)H]DA overflow in the NAc and PFC disappeared after 14 days withdrawal. The results show that chronic (-)nicotine differentially affects the NMDA-mediated [(3)H]DA release in the PFC and NAc of the rat.

Hexabromocyclododecane Inhibits Depolarization-Induced Increase in Intracellular Calcium Levels and Neurotransmitter Release in PC12 Cells

Environmental levels of the brominated flame retardant (BFR) hexabromocyclododecane (HBCD) have been increasing. HBCD has been shown to cause adverse effects on learning and behavior in mice, as well as on dopamine uptake in rat synaptosomes and synaptic vesicles. For other BFRs, alterations in the intracellular Ca(2+) homeostasis have been observed. Therefore, the aim of this study was to investigate whether the technical HBCD mixture and individual stereoisomers affect the intracellular Ca(2+) concentration ([Ca(2+)](i)) in a neuroendocrine in vitro model (PC12 cells). [Ca(2+)](i) and vesicular catecholamine release were measured using respectively single-cell Fura-2 imaging and amperometry. Exposure of PC12 cells to the technical HBCD mixture or individual stereoisomers did neither affect basal [Ca(2+)](i), nor the frequency of basal neurotransmitter release. However, exposure to HBCD (0-20 microM) did cause a dose-dependent reduction of a subsequent depolarization-evoked increase in [Ca(2+)](i). This effect was apparent only when HBCD was applied at least 5 min before depolarization (maximum effect after 20 min exposure). The effects of alpha- and beta-HBCD were comparable to that of the technical mixture, whereas the inhibitory effect of gamma-HBCD was larger. Using specific blockers of L-, N- or P/Q-type voltage-gated Ca(2+) channels (VGCCs) it was shown that the inhibitory effect of HBCD is not VGCC-specific. Additionally, the number of cells showing depolarization-evoked neurotransmitter release was markedly reduced following HBCD exposure. Summarizing, HBCD inhibits depolarization-evoked [Ca(2+)](i) and neurotransmitter release. As increasing HBCD levels should be anticipated, these findings justify additional efforts to establish an adequate exposure, hazard and risk assessment.

Kinetic characterization of l-[3H]glutamate uptake inhibition and increase oxidative damage induced by glutaric acid in striatal synaptosomes of rats

Glutaric acidemia type I (GA-I) is an inherited metabolic disease characterized by accumulation of glutaric acid (GA) and striatal degeneration. Although growing evidence suggests that excitotoxicity and oxidative stress play central role in the neuropathogenesis of this disease, mechanism underlying striatal damage in this disorder is not well established. Thus, we decided to investigate the in vitro effects of GA 10nM (a low concentration that can be present initial development this disorder) on l-[3H]glutamate uptake and reactive oxygen species (ROS) generation in synaptosomes from striatum of rats. GA reduced l-[3H]glutamate uptake in synaptosomes from 1 up to 30min after its addition. Furthermore, we also provided some evidence that GA competes with the glutamate transporter inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC), suggesting a possible interaction of GA with glutamate transporters on synaptosomes. Moreover, GA produced a significant decrease in the VMAX of l-[3H]glutamate uptake, but did not affect the KD value. Although the GA did not show oxidant activity per se, it increased the ROS generation in striatal synaptosomes. To evaluate the involvement of reactive species generation in the GA-induced l-[3H]glutamate uptake inhibition, trolox (0.3, 0.6 and 6μM) was added on the incubation medium. Statistical analysis showed that trolox did not decrease inhibition of GA-induced l-[3H]glutamate uptake, but decreased GA-induced reactive species formation in striatal synaptosomes (1, 3, 5, 10, 15 and 30min), suggesting that ROS generation appears to occur secondarily to glutamatergic overstimulation in this model of organic acidemia. Since GA induced DCFH oxidation increase, we evaluate the involvement of glutamate receptor antagonists in oxidative stress, showing that CNQX, but not MK-801, decreased the DCFH oxidation increase in striatal synaptosomes. Furthermore, the results presented in this report suggest that excitotoxicity elicited by low concentration of GA, could be in part by maintaining this excitatory neurotransmitter in the synaptic cleft by non-competitive inhibition of glutamate uptake. Thus the present data may explain, at least partly, initial striatal damage at birth, as evidenced by acute bilateral destruction of caudate and putamen observed in children with GA-I.