Protective effects of antioxidants on striatal dopamine release induced by organophosphorus pesticides.

Although the toxic effects of organophosphorus (OP) pesticides have been classically attributed to inhibition of the acetylcholinesterase, other neurotoxic mechanisms, as oxidative stress can also occur. Here we evaluated if antioxidants prevent the excessive dopamine release induced by OP pesticides in conscious and freely moving rats, using cerebral microdialysis technique. Intrastriatal infusion of paraoxon (5 mM), glufosinate (10 mM) or glyphosate (5 mM) significantly increased the dopamine release (1006 ± 106%, 991 ± 142%, and 1164 ± 128%, relative to baseline, respectively). To evaluate if these increased dopamine release could be related to oxidative stress, we pretreated animals with antioxidants glutathione (GSH, 400 or 800 µM), dithiothreitol (DTT, 5 or 10 µM), trolox (1 or 3 mM), and α-lipoic acid (ALA, 400 or 800 µM) before administration of OP pesticides. Intrastriatal administration of the antioxidants GSH, DTT, trolox, and ALA was highly effective in preventing the glyphosate and glufosinate-induced dopamine overflow. However, only GSH (800 µM) significantly decreased the effect of paraoxon on dopamine levels. The high toxicity of this pesticide and the low concentrations used could explain this lack of effect in our experimental conditions. The fact that ROS scavengers prevent the excessive dopamine release induced by OP pesticides, further supports the view that dopamine overflow can cause neuronal damage mediated, at least in part, by oxidative stress.

In vivo neurochemical characterization of clothianidin induced striatal dopamine release.

Clothianidin (CLO) is a neonicotinoid insecticide with selective action on nicotinic acetylcholine receptors. The aim of this study was to determine the neurochemical basis for CLO-induced striatal dopamine release using the microdialysis technique in freely moving and conscious rats. Intrastriatal administration of CLO (3.5mM), produced an increase in both spontaneous (2462 ± 627% with respect to basal values) and KCl-evoked (4672 ± 706% with respect to basal values) dopamine release. This effect was attenuated in Ca(2+)-free medium, and was prevented in reserpine pre-treated animals or in presence of tetrodotoxin (TTX). To investigate the involvement of dopamine transporter (DAT), the effect of CLO was observed in presence of nomifensine. The coadministration of CLO and nomifensine produced an additive effect on striatal dopamine release. The results suggest that the effect of CLO on striatal dopamine release is predominantly mediated by an exocytotic mechanism, Ca(2+), vesicular and TTX-dependent and not by a mechanism mediated by dopamine transporter.

Alterations of 3,4-dihydroxyphenylethylamine and its metabolite 3,4-dihydroxyphenylacetic produced in rat brain tissues after systemic administration of saxitoxin.

We have measured the dopamine levels in some discrete rat brain regions after acute intraperitoneal administration of saxitoxin (STX). STX is one of the several toxins that causes paralytic shellfish poisoning (PSP). PSP is a serious public health concern through the world. Certain dinoflagellates are able of producing STX, a powerful neurotoxic compound, which blocks the voltage sensitive sodium channels, entailing to the appearance of the main symptoms of poisoning by PSP: muscular paralysis and respiratory depression. The goal in this study was to analyze the effect of STX on dopamine levels in discrete rat brain regions after its acute intraperitoneal administration. Different experimental periods were analyzed for STX doses (5 and 10 µg kg(-1) body weight). With low dose, experimental periods were: 30, 60 and 120 min. With high dose, experimental period was just 30 min. At the end of each experimental period, animals were sacrificed by cervical dislocation. Brains were removed and dissected in: hypothalamus, striatum, midbrain, brain stem, right and left hemispheres. This is to our knowledge, the first report in which a sublethal dose of STX administered intraperitoneally results in an acute alteration of dopamine (DA) production and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC).

Behavioral and biochemical effects of neonicotinoid thiamethoxam on the cholinergic system in rats.

Thiamethoxam is a neonicotinoid insecticide, a group of pesticides that acts selectively on insect nicotinic acetylcholine receptors (nAChRs), with only a little action on mammalian nAChRs. Nevertheless, the selectivity of neonicotinoids for the insect nAChRs may change when these substances are metabolized. Therefore, we aimed to determine the potential effects of thiamethoxam on mammalian brain, testing the performance in the open field and elevated plus-maze of rats exposed to this insecticide and, in order to establish the neurochemical endpoints, we measured the acetylcholinesterase activity in different brain regions (hippocampus, striatum and cortex) and the high-affinity choline uptake (HACU) in synaptosomes from rat hippocampus. Treated animals received thiamethoxam (25, 50 or 100mg/kg) for 7 consecutive days. The results showed that treatment with thiamethoxam induced an increase in the anxiety behavior at two doses (50 or 100mg/kg). Moreover, there was a significant decrease in both HACU and acetylcholinesterase activity. Our hypothesis is that thiamethoxam (or its metabolites) could be acting on the central rats nAChRs. This would produce an alteration on the cholinergic transmission, modulating the anxiety behavior, acetylcholinesterase levels and HACU.

Evaluation of the effects and mechanisms of action of flutriafol, a triazole fungicide, on striatal dopamine release by using in vivo microdialysis in freely moving rats.

The purpose of the present work was to assess the effects of flutriafol, a triazole fungicide, on in vivo dopamine (DA) release from rat striatum, using brain microdialysis coupled to high-performance liquid chromatography with electrochemical detection (HPLC-EC). Intrastriatal administration of flutriafol (1, 6 and 12 mM) produced significant concentration-dependent increases in DA levels to 218.5+/-51%, 1376+/-245% and 3093+/-345% compared with basal values, respectively. Those increases in DA levels could be due to an increased DA exocytotic release and/or a change in the activity of DA transporter (DAT). Thus, we investigated the effects of flutriafol (6mM) under Ca(++)- or Na(+)-free conditions, and after pretreatment with reserpine and TTX. When flutriafol was perfused in either Ca(++)- or Na(+)-free Ringer, the DA levels reduced 92% and 70%, respectively; perfusion of flutriafol in TTX-treated (10 microM) or reserpine-pretreated animals (10mg/kg), reduced the levels of DA to 73% and 86%, respectively. Co-infusion of flutriafol and nomifensine (20 microM) shows that the flutriafol-induced DA release did not involve the DAT. Our results suggest that flutriafol induces DA release via vesicular-, Ca(++)-, Na(+)- and TTX-dependent mechanism, being independent of DAT.

Comparative effects of organic and inorganic mercury on in vivo dopamine release in freely moving rats

The present study was carried out in order to compare the effects of administration of organic (methylmercury, MeHg) and inorganic (mercury chloride, HgCl 2 ) forms of mercury on in vivo dopamine (DA) release from rat striatum. Experiments were performed in conscious and freely moving female adult Sprague-Dawley (230-280 g) rats using brain microdialysis coupled to HPLC with electrochemical detection. Perfusion of different concentrations of MeHg or HgCl 2 (2 muL/min for 1 h, N = 5-7/group) into the striatum produced significant increases in the levels of DA. Infusion of 40 muM, 400 muM, or 4 mM MeHg increased DA levels to 907 ± 31, 2324 ± 156, and 9032 ± 70 percent of basal levels, respectively. The same concentrations of HgCl 2 increased DA levels to 1240 ± 66, 2500 ± 424, and 2658 ± 337 percent of basal levels, respectively. These increases were associated with significant decreases in levels of dihydroxyphenylacetic acid and homovallinic acid. Intrastriatal administration of MeHg induced a sharp concentration-dependent increase in DA levels with a peak 30 min after injection, whereas HgCl 2 induced a gradual, lower (for 4 mM) and delayed increase in DA levels (75 min after the beginning of perfusion). Comparing the neurochemical profile of the two mercury derivatives to induce increases in DA levels, we observed that the time-course of these increases induced by both mercurials was different and the effect produced by HgCl 2 was not concentration-dependent (the effect was the same for the concentrations of 400 muM and 4 mM HgCl 2 ). These results indicate that HgCl 2 produces increases in extracellular DA levels by a mechanism differing from that of MeHg.

Comparative effects of organic and inorganic mercury on in vivo dopamine release in freely moving rats.

The present study was carried out in order to compare the effects of administration of organic (methylmercury, MeHg) and inorganic (mercury chloride, HgCl 2 ) forms of mercury on in vivo dopamine (DA) release from rat striatum. Experiments were performed in conscious and freely moving female adult Sprague-Dawley (230-280 g) rats using brain microdialysis coupled to HPLC with electrochemical detection. Perfusion of different concentrations of MeHg or HgCl 2 (2 microL/min for 1 h, N = 5-7/group) into the striatum produced significant increases in the levels of DA. Infusion of 40 microM, 400 microM, or 4 mM MeHg increased DA levels to 907 +/- 31, 2324 +/- 156, and 9032 +/- 70% of basal levels, respectively. The same concentrations of HgCl 2 increased DA levels to 1240 +/- 66, 2500 +/- 424, and 2658 +/- 337% of basal levels, respectively. These increases were associated with significant decreases in levels of dihydroxyphenylacetic acid and homovallinic acid. Intrastriatal administration of MeHg induced a sharp concentration-dependent increase in DA levels with a peak 30 min after injection, whereas HgCl 2 induced a gradual, lower (for 4 mM) and delayed increase in DA levels (75 min after the beginning of perfusion). Comparing the neurochemical profile of the two mercury derivatives to induce increases in DA levels, we observed that the time-course of these increases induced by both mercurials was different and the effect produced by HgCl 2 was not concentration-dependent (the effect was the same for the concentrations of 400 microM and 4 mM HgCl 2 ). These results indicate that HgCl 2 produces increases in extracellular DA levels by a mechanism differing from that of MeHg.

In vivo neurochemical characterization of anatoxin-a evoked dopamine release from striatum.

Anatoxin-a (AnTx) is a natural neurotoxin, which acts as a potent and stereoselective agonist at the nicotinic acetylcholine receptors. The in vivo actions of the AnTx on dopamine (DA) release are scarcely characterized. The aim of this study was to determine the neurochemical bases for AnTx-induced striatal DA release, using the brain microdialysis technique, in freely moving rats. Local application of AnTx (3.5 mM) through the microdialysis probe produced an increase in striatal DA levels (701 +/- 51% with respect to basal values). The effect of infusion of AnTx in Ca(2+)-free Ringer medium, in Na(+)-free Ringer medium and with TTX in the medium, was inhibited. Also, reserpine pre-treatment blocked the action of AnTx on striatal DA levels. To investigate the involvement of the DA transporter, the effects of AnTx were observed in the presence of nomifensine. The coadministration of AnTx and nomifensine evoked an additive effect on striatal DA levels. The latter results show that the DA release is not mediated by a decreased DA uptake. Taken as a whole, these results suggest that the effects of AnTx are predominantly mediated by an exocytotic mechanism, Ca(2+)-, Na(+)- and TTX-dependent, and not by a mechanism mediated by the DA transporter.

In vivo effects of the anatoxin-a on striatal dopamine release.

Anatoxin-a is an important neurotoxin that acts a potent nicotinic acetylcholine receptor agonist. This characteristic makes anatoxin-a an important tool for the study of nicotinic receptors. Anatoxin-a has been used extensively in vitro experiments, however anatoxin-a has never been studied by in vivo microdialysis studies. This study test the effect of anatoxin-a on striatal in vivo dopamine release by microdialysis.The results of this work show that anatoxin-a evoked dopamine release in a concentration-dependent way. Atropine had not any effect on dopamine release evoked by 3.5 mM anatoxin-a. However, perfusion of nicotinic antagonists mecamylamine and alpha-bungarotoxin induced a total inhibition of the striatal dopamine release. Perfusion of alpha7*-receptors antagonists, metillycaconitine or alpha-bungarotoxin, partially inhibits the release of dopamine stimulated by anatoxin-a. These results show that anatoxin-a can be used as an important nicotinic agonist in the study of nicotinic receptor by in vivo microdialysis technique and also support further in vivo evidences that alpha7*nicotinic AChRs are implicated in the regulation of striatal dopamine release.

Effects of manganese on extracellular levels of dopamine in rat striatum: an analysis in vivo by brain microdialysis.

The aim of this study is to determine the effects of intrastriatal administration of MnCl2, on the extracellular levels of dopamine (DA) and metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in basal conditions and stimulated by depolarization with KCl and pargyline administration. Also, we studied the effect of MnCl2 on extracellular levels of l-Dopa in the presence of aromatic amino acid decarboxylase (AADC) inhibitor 3-hydroxybencilhydracine-HCl (NSD 1015). This study concluded that MnCl2, reduced the basal and K+-stimulated DA-release in striatum, without notably affecting the DOPAC and HVA levels. Intraperitoneal injection of pargyline increased striatal DA levels, decreasing DOPAC and HVA levels. The infusion of MnCl2 removed the increase in DA levels, without affecting DOPAC and HVA levels. Perfusion of NSD 1015 increased the extracellular levels of L-DOPA in striatum, and MnCl2 increased the effect of NSD1015 on L-Dopa.

Protective effects of glutathione and cysteine on the methylmercury-induced striatal dopamine release in vivo.

The possible protective effects of glutathione (GSH), cysteine (CYS) and methionine (MET) on the Methylmercury (MeHg)-induced dopamine (DA) release from rat striatum were investigated using in vivo microdialysis coupled to HPLC with electrochemical detection. Intrastriatal infusion of MeHg 400 microM increased extracellular DA levels to 1941 +/- 199% in terms of basal levels. Infusion of MeHg 400 microM in GSH 400 microM pretreated animals, only increased striatal DA levels to 465 +/- 104%, in terms of basal levels, this increase being 76% lower than induced by MeHg alone. Conversely, the infusion of MeHg 400 microM after infusion of GSH 400 microM increased DA levels to 1019 +/- 96% in terms of basal levels, this increase being 47.5% lower than that observed in MeHg non-pretreated animals. The infusion of MeHg 400 microM in CYS 400 microM -pretreated animals, increased striatal DA levels to 740 +/- 149%, in terms of basal levels, this increase being 62% lower than that induced by MeHg in non-pretreated animals. The infusion of MeHg 400 microM in MET 400 microM pretreated animals increased striatal DA levels to 2011 +/- 230% in terms of basal, an increase that was not significantly different from that produced by MeHg 400 muM alone. In summary, the administration of compounds containing free -SH groups prevented the MeHg-induced DA release from rat striatum, probably due to the binding of MeHg to -SH groups. This would result in a lower metal availability to interact with -SH membrane proteins groups, which would decrease MeHg ability to interact with DA transporter.

Mechanisms underlying domoic acid-induced dopamine release from striatum: an in vivo microdialysis study.

The brain microdialysis technique has been used to examine the in vivo effects of the neurotoxin domoic acid (an ionotropic glutamate receptor agonist) on dopamine (DA) release in the striatum of conscious and freely moving rats. Local application of domoic acid (500 microM) through the microdialysis probe produced an increase in striatal DA content (597 +/- 96% with respect to basal levels). The release of DA induced by domoic acid was not attenuated in a Ca(+2)-free medium (469 +/- 59%) or after pretreatment with 10 mg/kg reserpine (533 +/- 79%). Intrastriatal infusion of 1 microM tetrodotoxin (TTX) partially reduced the domoic acid-evoked DA release (278 +/- 34%). Moreover, domoic acid perfusion had no effect on K+-evoked DA release. The results suggest that domoic acid increases the striatal DA release according to a reserpine-independent, calcium-independent and partially TTX-insensitive mechanism, suggesting that these effects probably involve a nonexocytotic process. On the other hand, the inhibitor of DA uptake nomifensine (10 microM) reduced the domoic acid-evoked DA release (356 +/- 59%), suggesting that a carrier-dependent mechanism could be involved in the effect of domoic acid on the striatal DA levels.

Effects of successive intrastriatal methylmercury administrations on dopaminergic system.

The present study was carried out in order to determine the effects of intrastriatal administration of different doses (40 microM, 400 microM, and 4mM) of methylmercury (MeHg) on dopaminergic system of rat striatum. Experiments were performed in conscious and freely moving rats using brain microdialysis coupled with liquid chromatography. Intrastriatal administration of MeHg produced significant increases in dopamine (DA) striatal levels (907+/-7%, 1870+/-319%, and 7971+/-534% for the doses of 40, 400 microM, and 4mM, with respect to basal). The increase in DA levels was associated with significant decreases in extracellular levels of its main metabolites dihydroxyphenylacetic acid (DOPAC) and homovallinic acid (HVA) (65.0+/-3.0% and 52.2+/-1.3%, respectively) using the dose of 4mM MeHg, whereas nonsignificant changes in metabolite levels were observed with the doses of 40 and 400 microM MeHg. A second infusion of 4mM MeHg 24h after first infusion also produced a rise of DA levels, but this increase was very small as compared with that produced by first infusion (7971+/-534% versus 985+/-186%). This second infusion of 4mM MeHg also decreased DOPAC and HVA levels, but this decrease was not significant as compared with that observed after first infusion (65.0+/-3.0% and 52.2+/-1.3% versus 62.4+/-5.2% and 63.4+/-7.4%, respectively). We discuss these effects based on a stimulated DA release and/or a decreased DA intraneuronal degradation.

Protection of methylmercury effects on the in vivo dopamine release by NMDA receptor antagonists and nitric oxide synthase inhibitors.

The possible protective effects of NMDA receptor antagonists dizocilpine (MK-801) and D(-)-2-amino-5-phosphonopentanoic acid (AP5), and nitric oxide synthase (NOS) inhibitors L-nitro-arginine methyl ester (L-NAME) and 7-nitro-indazol (7-NI) on the methylmercury (MeHg)-induced dopamine (DA) release from rat striatum were investigated using in vivo microdialysis. Intrastriatal infusion of 400 microM or 4 mM MeHg increased the extracellular DA levels to 1941+/-199 and 7971+/-534% with respect to basal levels. Infusion of 400 microM or 4 mM MeHg in 400 microM MK-801 pretreated animals, increased striatal DA levels to 677+/-126 and 2926+/-254%, with respect to basal levels, these increases being 65 and 63% smaller than those induced by MeHg in non-pretreated animals. Infusion of 400 microM or 4 mM MeHg in 400 microM AP5 pretreated animals, increased striatal DA levels to 950+/-234 and 2251+/-254% with respect to basal levels, these increases being 51 and 72% smaller than those induced by MeHg in non-pretreated animals. Infusion of 400 microM MeHg in 100 microM L-NAME or 7-NI pretreated animals, increased the extracellular DA levels to 1159+/-90 and 981+/-292%, with respect to basal levels, these increases being 40 and 50% smaller than those induced by MeHg in non-pretreated animals. In summary, MeHg acts, at last in part, through an overstimulation of NMDA receptors with possible NO production to induce DA release, and administration of NMDA receptor antagonists and NOS inhibitors protects against MeHg-induced DA release from rat striatum.

Mechanism of action of methylmercury on in vivo striatal dopamine release. Possible involvement of dopamine transporter.

Methylmercury (MeHg) produces significant increases in the spontaneous output of dopamine (DA) from rat striatal tissue. The mechanism through MeHg produces such increase in the extracellular DA levels could be due to increased DA release or decreased DA uptake into DA terminals. One of the aims of this study was to investigate the role of DA transporter (DAT) in the MeHg-induced DA release. Coinfusion of 400 microM MeHg and nomifensine (50 microM) or amphetamine (50 microM) produced increases in the release of DA similar to those produced by nomifensine and amphetamine alone. In the same way, MeHg-induced DA release was not attenuated under Ca(2+)-free conditions or after pretreatment with reserpine (10 mg/kg i.p.) or tetrodotoxin (TTX), suggesting that the DA release was independent of calcium and vesicular stores, as well as it was not affected by the blockade of voltage sensitive sodium channels. Thus, to investigate whether depolarization of dopaminergic terminal was able to affect MeHg-induced DA release, we infused 75 mM KCl through the dialysis membrane. Our results clearly showed a decrease induced by MeHg in the KCl-evoked DA release. Taken together, these results suggest that MeHg induces release of DA via transporter-dependent, calcium- and vesicular-independent mechanism and it decreases the KCl-evoked DA release.

Localization of NADPH diaphorase activity in the human visual cortex

The present report describes the activity of NADPH-diaphorase (NADPHd) in area 17 of autopsied normal human visual cortex. Four human brains from autopsy tissue (4-8 h postmortem) were fixed by immersion in 4 per cent paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2-7.4, or in 10 per cent formalin for 24 h. NADPHd histochemistry was done using the malic enzyme indirect method. The neurpile pattern of enzyme activity presented a clear six layer appearance. Cell morphology and the laminar distribution of 73 NADPHd-positive neurons are descrived. All neurons found in area 17 of human cortex were sparsely spiny or smooth cells, located in all cortical layers exept layer 4c. Quantitative analysis of the branching pattern of the dendritic tree was carried out. A symmetrical pattern was observed with no particular dendritic bias except for a few white matter and layer 1 cells. Larger dendritic fields were found in white matter cells when compared to the other corical layers. Comparison of cell densities for gray and white matters showed that 85 per cent of the NADPHd-positive neurons were located in the white matter. NADPH was colocalized with nitric oxide synthase which produces nitric oxide, a short-life neuromediator implicated in synaptic plasticity, neuroprotection, and neurotoxicity. thus, the spatial distribution of the NADPHd cells is important for posterior functional studies of the neuromediators in the brain