Synaptosomal GABA uptake decreases in paraoxon-treated rat brain

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Toxicology 244 (2008) 42–48

Synaptosomal GABA uptake decreases in paraoxon-treated rat brain Moslem Mohammadi a,∗ , Esmaeel Ghani a , Asghar Ghasemi b , Ali Khoshbaten c , Alireza Asgari c a

c

Department of Physiology and Biophysics, Baqiyatallah University of Medical Sciences, Tehran, Iran b Prevention of Metabolic Disorders Research Center, Research Institute for Endocrine Sciences, Shaheed Beheshti University of Medical Sciences, Tehran, Iran Research Center for Chemical Injuries (RCCI), Baqiyatallah University of Medical Sciences, Tehran, Iran Received 22 September 2007; received in revised form 25 October 2007; accepted 25 October 2007 Available online 4 November 2007

Abstract A synaptosomal model was used to evaluate in vivo effects of paraoxon on the uptake of [3 H]GABA in rat cerebral cortex and hippocampus. Male Wistar rats were given a single intraperitoneal injection of one of three doses of paraoxon (0.1, 0.3, or 0.7 mg/kg) and acetylcholinesterase (AChE) activity in the plasma, cerebral cortex, and hippocampus was measured at 30 min, 4 h, and 18 h after exposure. [3 H]GABA uptake in synaptosomes was also studied in another series of animals. Paraoxon administration (0.3 and 0.7 mg/kg) caused significant inhibition of AChE activity in the plasma and both brain areas at all time points. 0.1 mg/kg paraoxon significantly inhibited AChE activity but only in the plasma for 4 h, the activity was completely recovered at 18 h. GABA uptake was significantly (p < 0.001) reduced in both cerebral cortex (18–32%) and hippocampal (16–23%) synaptosomes at all three time points after administering 0.7 mg/kg of paraoxon, a dose that seems to be sufficient to induce seizure activity. l-DABA, an inhibitor of neuronal GABA transporter, allowed us to conclude that the uptake was mediated primarily by neuronal GABA transporter GAT-1. In conclusion, present data suggests that GABA uptake by synaptosomes decreases probably secondary to paraoxon-induced seizure activity. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Paraoxon; Synaptosome; GABA uptake; Seizure

1. Introduction Paraoxon (the neurotoxic metabolite of organophosphorus (OP) insecticide parathion) exerts acute toxicity in target organisms by inhibition of acetylcholinesterase (AChE), leading to the accumulation of acetylcholine in ∗ Corresponding author at: Department of Physiology and Biophysics, Baqiyatallah University of Medical Sciences, P.O. box 19395-6538, Tehran, Iran. Tel.: +98 21 22281561; fax: +98 21 22281561. E-mail address: [email protected] (M. Mohammadi).

cholinergic synapses and overstimulation of the cholinergic system (Rocha et al., 1996). The major concern of OP poisoning is the convulsive activity that is produced in susceptible areas as a result of elevated ACh. Convulsive activity develops almost immediately after exposure to OP and progresses rapidly to status epilepticus, causing profound neuronal damage (Shih and McDonough, 1997). While central muscarinic mechanisms appear to be responsible for the initiation of convulsions, other neurotransmitter systems may be involved in the propagation or maintenance of seizures (McDonough and Shih, 1997). It has been suggested that

0300-483X/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2007.10.024

M. Mohammadi et al. / Toxicology 244 (2008) 42–48

␥-aminobutyric acid (GABA) would be implicated in the convulsant syndrome induced by OP compounds, because GABA agonists and benzodiazepines are able to inhibit OP-induced seizures (Shih et al., 1991; Rump and Kowalczyk, 2004). Changes in the levels and function of inhibitory amino acid GABA during OP-induced seizures have been controversial. One group has reported that brain GABA levels were reduced in animals that convulsed during intoxication with paraoxon (Karr and Matin, 1972). In contrast, other studies found no change (Coudray-Lucas et al., 1984; Lallement et al., 1991) and even increases (Fosbraey et al., 1990) in the levels of GABA after intoxication with OP compounds. Studies on GABAergic transmission in OPs intoxication have focused primarily on analysis of total GABA levels in brain homogenates (Coudray-Lucas et al., 1984; Liu et al., 1988; Fosbraey et al., 1990), brain slices (Santos et al., 2002), cultured neurons (Rocha et al., 1996), and after microdialysis (Lallement et al., 1991). Efficient reuptake mechanism of GABA into presynaptic neurons or glial cells would contribute to modify the extracellular GABA levels (Dalby, 2003). Two more recent in vitro studies were carried out in our laboratory indicating that paraoxon has an inhibitory effect on the uptake of [3 H]GABA by rat cerebellar (Shahroukhi et al., 2007), and cortical (Ghasemi et al., 2007) isolated nerve terminals (synaptosomes); worth it to mention that the synaptosomes were prepared from untreated rats. A question that need be answered is whether the synaptosomes prepared from paraoxon-treated rats behave as like and demonstrate similar changes. Therefore, in present study, we used cortical and hippocampal synaptosomes from paraoxon-treated rats to detect changes in nerve terminal GABA uptake.

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food and water ad libitum. Paraoxon was dissolved in corn oil and dilutions were carefully made in such a way to be administered at 1 ml/kg body weight. Animals were given a single injection of one of three doses of paraoxon (0.1, 0.3, or 0.7 mg/kg, i.p.) and were killed after dosing by decapitation at three time points (30 min, 4 h and 18 h). Control animals were injected with corn oil at the same rate. At each group, seven rats were utilized to assay AChE. [3 H]GABA uptake was determined in another series of animals (5–7 rats/group). All procedures were in accordance with the standards for animal care established by the Ethical Committee of the Baqiyatallah University of Medical Sciences. 2.3. AChE assay AChE activity was determined using the modified method of Ellman et al. (1961). Briefly, rats were anesthetized with diethyl ether and enough blood obtained by cardiac puncture in heparinized syringes (Pope et al., 1991). Rats were then decapitated, brain removed quickly and placed immediately in ice-cold 0.32 M sucrose buffered with phosphate (0.1 M, pH 7.4) to obtain two hippocampus and cerebral cortices. When dissected, tissues were weighed and homogenized in 10 volumes of phosphate buffer (0.1 M, pH 7.4) containing 1% Triton X-100 and 1 M NaCl. Whole heparinized blood was centrifuged to separate plasma. The incubation mixture in a total volume of 2 ml consisted of: 1.8 ml of 5,5 -dithiobis(2nitrobenzoic) acid (DTNB; 0.423 mM) in 0.1 M phosphate buffer (pH 7.4), 0.1 ml of homogenate (10 mg/ml) or diluted plasma (1:10), and 0.1 ml of acetylthiocholine iodide (ATC, final concentration 1 mM). The absorbance at 412 nm was read immediately after addition of substrate (ATC) and measured during the following 5 min at room temperature. Specific activity in plasma was calculated as nanomoles substrate hydrolyzed per min per ml, whereas activity in brain regions was calculated as nanomoles substrate hydrolyzed per min per mg protein.

2. Materials and methods 2.1. Chemicals [3 H]GABA (86 Ci/mmol) was purchased from Amersham Bioscience UK. Paraoxon [o,o -diethyl-p-nitrophenyl phosphate; 90% pure], nipecotic acid, aminooxyacetic acid (AOAA), ␥-amino-n-butyric acid (GABA), ␤-alnine, ldiaminobutyric acid (l-DABA), and acetylthiocholine iodide (ATC) were obtained from Sigma Chemical Co., Germany. 5,5 -Dithiobis(2-nitrobenzoic)acid (DTNB) and bovine serum albumin (BSA) were prepared from Fluka (Swiss). Other materials prepared from Merck Company, Germany. 2.2. Animals and treatment Adult male Wistar rats (200–270 g) were kept under standard laboratory conditions, with a 12 h light/dark cycle and

2.4. Preparation of synaptosomes After decapitation, samples of cerebral cortex and hippocampus were dissected, weighed, and homogenized in 10 volumes of ice-cold 0.32 M sucrose buffered at pH 7.4 with phosphate (final concentration 0.1 M). A synaptosomal preparation was isolated according to the method of Raiteri et al. (2003) with minor modifications. Briefly, the homogenates were centrifuged at 1000 × g for 5 min (4 ◦ C), to remove nuclei and cellular debris. The synaptosomal fractions were isolated from supernatants by centrifugation at 12,000 × g for 20 min. The synaptosomal pellets were then resuspended in a physiological medium having the following composition (mM): NaCl, 125; KCl, 3; MgSO4 , 12; NaH2 PO4 , 1; NaHCO3 , 22; Glucose, 10 (aeration with 95% O2 and 5% CO2 ); pH 7.4. Protein concentration was adjusted at 1 mg/ml.

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Table 1 AChE activity in the plasma of rats following exposure to three doses of paraoxon Time

30 min 4h 18 h

Control

260 ± 5.9 259.7 ± 6.4 261.7 ± 5.8

Paraoxon 0.1 mg/kg

0.3 mg/kg

0.7 mg/kg

231.2 ± 3.4* A (11.1%) 238.4 ± 3.6* A (8.2%) 256.8 ± 4.9 B (1.9%)

101.9 ± 5.3** A (60.8%) 146.7 ± 6** B (43.5%) 187.3 ± 5.1** C (28.4%)

40.2 ± 1.9**A (84.6%) 83.4 ± 2.5** B (67.9%) 151.3 ± 3.2** C (42.2%)

AChE activity is expressed as nmoles substrate hydrolyzed per min per ml plasma. Values are given as mean ± S.E.M. (n = 7 rats/group). Number in parenthesis indicates percent inhibition of AChE activity. *p < 0.05, **p < 0.001 compared with the respective control groups. Means within a column not followed by the same letter are significantly different (p < 0.01).

between the plasma and either cerebral cortex or hippocampus AChE activity. p < 0.05 was considered significant.

2.5. Protein assay Protein content of homogenate and synaptosomal fractions was determined by the method of Bradford (1976) and BSA was used as standard.

3. Results Changes reported below occurred at all three time points unless stated otherwise.

2.6. [3 H]GABA uptake assay Synaptosomes were incubated at 37 ◦ C. Uptake was initiated by the addition of a mixture of cold and tritiated GABA (400 nM, 1.5% of which was tritiated) and lasted for 10 min. 10 ␮M AOAA (a GABA transaminase inhibitor) was used in all experiments to prevent GABA metabolism. 50 mM nipecotic acid (a GABA uptake inhibitor), 500 ␮M l-DABA (a neuronal GABA transporter, GAT, inhibitor), and 100 ␮M ␤-alanine (a glial GABA transporter inhibitor), where included, were preincubated with the synaptosomes 15 min before the initiation of uptake. Reaction was stopped by adding 1 ml of cold saline after 10 min. Synaptosomes were centrifuged for 10 min at 10,000 × g twice and plate was solubilized in 1% sodium dodecyl sulfate. Scintillator was added to each sample and their radioactivities were counted with liquid scintillation counter (Betamatic, Contron, France). 2.7. Statistical analysis The results were presented as mean ± S.E.M. Comparison of GABA uptake and AChE activity was done by paired t-test and one-way analysis of variance (ANOVA), and if ANOVA results were significant followed by post hoc Tukey test for comparison. Pearson correlation coefficients were calculated

3.1. Signs of toxicity Within 30 min following exposure, overt signs of cholinergic crisis (i.e., lacrimation, salivation, urination, diarrhea, chewing, fasciculation, and tremor) were observed in rats exposed to 0.7 mg/kg paraoxon. The two lower dose groups (0.1 and 0.3 mg/kg groups) expressed no clear signs of toxicity. However, all signs of paraoxon toxicity had subsided after 18 h. 3.2. Effect of paraoxon on AChE activity Plasma AChE activity was significantly inhibited in a dose-dependent manner for 4 h in all paraoxontreated groups. Activity was completely recovered at 18 h in animals exposed to 0.1 mg/kg paraoxon, and 4 and 18 h after paraoxon injection (0.3 and 0.7 mg/kg) a significant spontaneous reactivation was observed in the plasma AChE activity (Table 1). Paraoxon administration (0.3 or 0.7 mg/kg) caused a dose-dependent

Table 2 AChE activity in the cerebral cortex of rats following exposure to three doses of paraoxon Time

30 min 4h 18 h

Control

97.1 ± 2.2 96 ± 2.1 98.7 ± 2.1

Paraoxon (mg/kg) 0.1

0.3

0.7

91.1 ± 2 (6.2%) 92.2 ± 2.1 (3.9%) 95.4 ± 1.3 (3.3%)

57.4 ± 1.5** (40.9%) 59.7 ± 1.7** (37.8%) 62.1 ± 1.2** (37.1%)

15.5 ± 1.1** A (84%) 18.4 ± 0.6** A (80.8%) 34.5 ± 0.8** B (65%)

AChE activity is expressed as nmoles substrate hydrolyzed per min per mg protein. Values are given as mean ± S.E.M. (n = 7 rats/group). Number in parenthesis indicates percent inhibition of AChE activity. **p < 0.001 compared with the respective control groups. Means within a column not followed by the same letter are significantly different (p < 0.01).

M. Mohammadi et al. / Toxicology 244 (2008) 42–48

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Table 3 AChE activity in the hippocampus of rats following exposure to three doses of paraoxon Time

30 min 4h 18 h

Control

Paraoxon (mg/kg)

65.7 ± 2.3 66.3 ± 2.3 65.6 ± 2

0.1

0.3

0.7

60.8 ± 0.96 (7.5%) 62.6 ± 1.1 (5.6%) 64.8 ± 1.5 (1.2%)

38.2 ± 1.2** (41.9%) 41 ± 2.7** (38.2%) 44.5 ± 1.7** (32.2%)

11 ± 1.1** A (83.3%) 14.4 ± 0.74** A (78.3%) 23.4 ± 1.8** B (64.3%)

AChE activity is expressed as nmoles substrate hydrolyzed per min per mg protein. Values are given as mean ± S.E.M. (n = 7 rats/group). Number in parenthesis indicates percent inhibition of AChE activity. **p < 0.001 compared with the respective control groups. Means within a column not followed by the same letter are significantly different (p < 0.01).

significant inhibition of AChE activity in both cerebral cortex (Table 2) and hippocampus (Table 3). 18 h after 0.7 mg/kg paraoxon, activity was recovered (p < 0.01) in both brain areas (about 20%). Plasma AChE activity correlated significantly with both cerebral cortex and hippocampus AChE activity in rats treated with 0.3 or 0.7 mg/kg paraoxon (Table 4).

On the other hand, the GAT-1 selective inhibitor lDABA (500 ␮M) abolished approximately 90% of the carrier mediated GABA uptake in both brain areas in all groups; results for control and 0.7 mg/kg paraoxon groups were shown in Fig. 2A and B.

3.3. Effect of paraoxon on synaptosomal [3 H]GABA uptake

In the present study, we observed GABA uptake was reduced in both cerebral cortex and hippocampus after administering 0.7 mg/kg of paraoxon, a dose that seems to be sufficient to induce seizure activity. Brain cortex and hippocampus were selected in this work for synaptosomal preparations for the following

Non-carrier-mediated uptake was measured in paired tubes containing 50 mM nipecotic acid and was subtracted from total uptake to give the specific uptake. Typically, more than 98% of cortical and hippocampal uptake were prevented by nipecotic acid. All values are given as specific uptake and expressed as pmol/mg protein/min. Significant inhibition of [3 H]GABA uptake was noted in both cerebral cortex (Fig. 1A), and hippocampal (Fig. 1B) synaptosomes after paraoxon (0.7 mg/kg) exposure (p < 0.001).

4. Discussion

3.4. Inhibitor sensitivity of GABA transport ␤-Alanine (100 ␮M) produced no significant changes in the amount of GABA uptake by cortical and hippocampal synaptosomes in all groups (data not shown). Table 4 Correlation of AChE activity in plasma with AChE activity in cortex and hippocampus Paraoxon (mg/kg)

0.1 0.3 0.7

Correlation coefficient Plasma vs. cortex

Plasma vs. hippocampus

0.181 0.487* 0.955**

0.239 0.591** 0.845**

Correlation coefficients (r values) were determined by plotting residual plasma AChE activity vs. residual cortex or hippocampus AChE activity after treatment with three doses of paraoxon between 30 min and 18 h (n = 21 rats/dose). *p < 0.05, **p < 0.001.

Fig. 1. [3 H]GABA uptake by (A) cortical, and (B) hippocampal synaptosomes of rats following exposure to three doses of paraoxon. Values are given as mean ± S.E.M. (n = 5–7 rats/group). **p < 0.001 compared with the respective control groups. # Significant (p < 0.001) time effect within the same treatment group.

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Fig. 2. The effect of l-DABA (500 ␮M) on [3 H]GABA uptake in (A) cortical, and (B) hippocampal synaptosomes. In the presence of l-DABA, the uptake was significantly (p < 0.001) decreased by cortical and hippocampal synaptosomes in both control and 0.7 mg/kg paraoxon groups at all time points (significancy was not shown). Values are given as mean ± S.E.M. (n = 5 rats/group).

reasons: (1) hippocampus is known to be important in the initiation and maintenance of epileptic seizures (Costa et al., 2004; Harrison et al., 2004); (2) long-lasting excitability spread to cortical areas (Costa et al., 2004); (3) these areas are rich in cholinergic nerve terminals and are damaged after OP-induced seizures (Harrison et al., 2004); (4) high levels of GAT-1 mRNA have been found along brain cortex and hippocampus (Cecchini et al., 2004; Dalby, 2003). The systemic toxicity of OPs reflects the symptoms related to cholinergic hyperstimulation consequent to the irreversible loss of AChE catalytic activity, which typically emerges when inhibition exceeds 70% (Clegg and van Gemert, 1999). In the current study symptoms of paraoxon toxicity were observed only in the 0.7 mg/kg group, in which more than 80% of brain AChE activity was inhibited 30 min after exposure. All symptoms of paraoxon toxicity had subsided 18 h following the treatment in this group, at the time point when inhibition of AChE activity was less than 65%. Plasma AChE activity decreased and recovered more rapidly than it did in brain tissues. This rapid recovery of plasma AChE activity has been reported by other investigators (Pope et al., 1991; Padilla et al., 1994). It is thought that the

plasma AChE activity regenerates quickly because it is continually resynthesized by the liver. The strongest correlation in the plasma AChE activity with AChE activity in the cerebral cortex (r = 0.955) and hippocampus (r = 0.845) was obtained after treatment with 0.7 mg/kg paraoxon (Table 4). These data are in agreement with other studies (Pope et al., 1991; Padilla et al., 1994) suggesting that a recognized and accessible biomarker of exposure, circulating AChE activity, can be used to predict the extent of brain AChE inhibition following OP exposure under certain circumstances. While muscarinic cholinergic mechanisms are primarily involved in eliciting convulsions after an OP compound exposure, several neurotransmitter systems may be involved in the propagation and maintenance of OP-induced seizures (McDonough and Shih, 1997). GABA is the major inhibitory neurotransmitter in the central nervous system (CNS) and as such plays a key role in modulating neuronal activity (Bettler et al., 2004). The magnitude and duration of GABA synaptic action are regulated by plasma membrane proteins, termed GABA transporters (GATs), which mediate a high-affinity Na+ /Cl− dependent uptake of GABA into presynaptic axon terminals and glial processes (Minelli et al., 1996). Three subtypes of GATs, designated GAT1, GAT-2, and GAT-3, have been identified in the rat and human brain (Ueda and Willmore, 2000). GAT-1 and GAT-3 are the most likely candidates for regulating GABA transport in the brain. Neurons only express GAT-1 whereas GAT-3 is expressed by astrocytes (Dalby, 2003). GABA has been reported to be related to OP-induced toxicities, especially convulsions (Liu et al., 1988). Alterations in GABA levels after OP exposure are controversial. Despite findings that convulsion/seizure elicited by OP compounds may be due to a reduction in brain GABA levels (Karr and Matin, 1972; Shih et al., 1991), several studies have shown that changes in the guinea pig and rat brain GABA levels or metabolism are indeed secondary and in part compensatory action in response to soman-induced seizures (Fosbraey et al., 1990; McDonough and Shih, 1997). In this study, significant decrease of [3 H]GABA uptake was found in cortical (18–32%) and hippocampal (16–23%) synaptosomes only in 0.7 mg/kg group at all three time points studied (Fig. 1A and B). Although seizure activity was not assessed in this study by electroencephalographic (EEG) recordings, but it is well known that seizure will occur in rodents only when the brain AChE inhibition is over 65% (Tonduli et al., 2001). In the present study, in both brain areas, only 0.7 mg/kg of paraoxon inhibited AChE activity over this threshold (>80% inhibition) after 30 min

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(Tables 2 and 3), therefore it can be suggested that only in this group seizures could occur. Since GABA uptake did not change in other two paraoxon-treated groups, it may be concluded that decrement of GABA uptake is seizurerelated. The observation that changes in GABAergic system lagged behind paraoxon-induced seizure activity in our investigation parallel those reported by Fosbraey et al. (1990), and McDonough and Shih (1997). Decreases in GABA uptake have also been demonstrated in the thalamic synaptosomes from a rat model of epilepsy, genetic absence epilepsy rats from Strasbourg, (Sutch et al., 1999), and in dentate gyrus of kinate-induced epileptic rats (Patrylo et al., 2001). The results of the present study differ from those obtained by Coudray-Lucas et al., 1984 reporting paraoxon (1 mg/kg) did not significantly affect GABA levels in rat brain areas 4 h and 18 h after injection intraperitoneally. It should be noted that they measured GABA levels using homogenate technique, which measure both intracellular and extracellular levels of GABA. Furthermore, a microdialysis-based GABA measurement showed no change in extracellular GABA levels in the rat hippocampus during the first 30 min of soman-induced seizures (Lallement et al., 1991). In an electrophysiological in vitro study, paraoxon (0.3 ␮M) significantly increased the frequency of GABA-mediated miniature postsynaptic currents in hippocampal neuron cultures, implying that paraoxon at these low concentrations can affect GABAergic transmission (Rocha et al., 1996). The blockade of the GABA-A receptor in the above-mentioned study was observed by ≥3 ␮M paraoxon. Although we did not come across a report claiming how in vitro concentrations of drugs can give a clue of what the comparable in vivo concentration might be, or vice versa, there are reports in agreement with the present findings, indicating that paraoxon decreases [3 H]GABA uptake by rat cerebellum (Shahroukhi et al., 2007) and cerebral cortex (Ghasemi et al., 2007) at micromolar and millimolar concentrations, respectively. A direct extrapolation of our findings to paraoxon concentrations that used in the mentioned in vitro studies is important. Decreases in the synaptosomal uptake observed in their study (Ghasemi et al., 2007) by millimolar concentrations of paraoxon, seems less likely to occur in vivo. Although paraoxon concentrations in the brain were not measured in our study, but it seems that paraoxon in the brain may reach levels on the order of 10 ␮M during clinical intoxication (Rocha et al., 1996). Moreover, tabun decreased cerebral cortex GABA uptake at 1–2 mM concentrations in guinea pig (Szilagyi et al., 1993).

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The decrease in GABA uptake, presumably due to a change in the GAT system, may represent a compensatory response modulating neuronal overexcitation (Conti et al., 2004). At GABAergic synapses, acute inhibition of transporters prolongs postsynaptic GABA-A and GABA-B inhibitory currents, and spillover of extracellular GABA inhibits glutamate release from nearby excitatory terminals. However, transporter reversal can also occur under several conditions, including elevations in extracellular K+ concentration, which also occur during seizures. Thus, GABA transporter could contribute to seizure termination and propagation through heterotransport (Patrylo et al., 2001). ␤-Alanine is a weak inhibitor of GAT-1, whereas it can inhibit the rat uptake proteins GAT-2 and GAT-3 by 60–90% at a concentration of 100 ␮M (Sutch et al., 1999). l-DABA, on the other hand, is a strong inhibitor of neuronal GABA transporter GAT-1 (Wonnemann et al., 2000). Since most of synaptosomal GABA uptake was blocked by the neuronal uptake inhibitor l-DABA (Fig. 2A and B), it can be concluded that the uptake was primarily due to a neuronal carrier, GAT-1 (Wonnemann et al., 2000; Ghasemi et al., 2007) and not related to glial uptake. In conclusion, the data presented herein indicate that decrease in GABA uptake presumably is secondary to paraoxon-induced seizure activity. Acknowledgment This work was financially supported by Research Center for Chemical Injuries (RCCI) of Baqiyatallah University of Medical Sciences. References Bettler, B., Kaupmann, K., Mosbacher, J., Gassmann, M., 2004. Molecular structure and physiological functions of GABAB receptors. Physiol. Rev. 84, 835–867. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Cecchini, A.L., Soares, A.M., Giglio, J.R., Amara, S., Arantes, E.C., 2004. Inhibition of l-glutamate and GABA synaptosome uptake by crotoxin, the major neurotoxin from crotalus durissus terrificus venum. J. Venom Anim. Toxins. Incl. Trop. Dis. 10 (3), 260–279. Clegg, D.J., van Gemert, M., 1999. Determination of the reference dose for chlorpyrifos: preceedings of an expert panel. J. Toxicol. Environ. Health 2, 211–255. Conti, F., Minelli, A., Melone, M., 2004. GABA transporters in the mammalian cerebral cortex: localization, development and pathological implications. Brain Res. Rev. 45, 196–212. Costa, M.S., Rocha, J.B.T., Perosa, S.R., Cavalheiro, E.A., NaffahMazzacoratti, M.G., 2004. Pilocarpine-induced status epilepticus

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