Toward development of an in vitro model of methamphetamine-induced dopamine nerve terminal toxicity.
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To develop an in vitro model of methamphetamine (METH)-induced dopamine (DA) neurotoxicity, striatal synaptosomes were incubated at 37 degrees C with METH for different periods of time (10-80 min), washed once, then tested for DA transporter function at 37 degrees C. METH produced time- and dose-dependent reductions in the V(max) of DA uptake, without producing any change in K(m). Incubation of synaptosomes with the DA neurotoxins 1-methyl-4-phenyl-pyridinium ion, 6-hydroxydopamine, and amphetamine under similar conditions produced comparable effects. In contrast, incubation with fenfluramine, a serotonin neurotoxin, did not. METH-induced decreases in DA uptake were selective, insofar as striatal glutamate uptake was unaffected. Various DA transporter blockers (cocaine, methylphenidate, and bupropion) afforded complete protection against METH-induced decreases in DA uptake, without producing any effect themselves. METH's effects were also temperature dependent, with greater decreases in DA uptake occurring at higher temperatures. Tests for residual drug revealed small amounts (0.1-0.2 microM) of remaining METH, but kinetic studies indicated that decreases in DA uptake were not likely to be due to METH acting as a competitive inhibitor of DA uptake. Decreases in the V(max) of DA uptake were not accompanied by decreases in B(max) of [(3)H]WIN 35,428 binding, possibly because there is no mechanism for removing damaged DA nerve endings from the in vitro preparation Collectively, these results give good support to the development of a valid in vitro model that may prove helpful for elucidating the mechanisms underlying METH-induced DA neurotoxicity. (+info)
The absence of impairment of cliff avoidance reaction induced by subchronic methamphetamine treatment in inbred strains of mice.
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Cliff avoidance reaction (CAR), an index of behavioral teratology in rodents, can be impaired by motor, arousal, or cognitive dysfunction. We formerly reported subchronic administration of methamphetamine (MAP) induced the CAR impairment, which might reflect MAP-induced cognitive dysfunction, in three strains of rats. In this study, the effects of subchronic MAP treatment on the behavioral sensitization in stereotypy (stereotypy sensitization) and CAR were examined in two inbred strains of male mice; C57BL/6J(C57) and DBA/2J(DBA). The animals received 4 mg/kg/day MAP intraperitoneally for 28 days. There were apparent strain differences in the development of stereotypy sensitization induced by chronic MAP; DBA mice developed stereotypy sensitization quickly, but C57 did not. Unlike rats, neither strains of mice showed the CAR impairment. These results suggest that chronic MAP (4 mg/kg) administration did not introduce any cognitive dysfunction measured by CAR in the two inbred mice, DBA and C57. The discrepancy between rats and mice is still unclear. It might relate to the species-selective effect of MAP on the CAR impairment. Further studies should to be required. (+info)
Effects of flunarizine on dopamine dependent behaviours in rats.
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1. Radio-ligand binding study has demonstrated that flunarizine has a high affinity for the rat striatal D 2 dopamine (DA) receptors. 2. In the present behavioural study conducted in rats it was observed that flunarizine, unlike the postsynaptic striatal D 2 DA receptor agonist apomorphine, did not induce stereotyped behaviour (SB) in rats. This indicates that flunarizine does not act as an agonist at the postsynaptic striatal D 2 DA receptors. 3. Flunarizine however, like the postsynaptic striatal D 2 DA receptor antagonist haloperiodal, inhibited the conditioned avoidance response, induced catalepsy and antagonized the SB induced by the DA agonists apomorphine and methamphetamine. 4. Our findings indicate that flunarizine acts as a postsynaptic striatal D 2 DA receptor antagonist. (+info)
Regulation of phosphorylation of the GluR1 AMPA receptor in the neostriatum by dopamine and psychostimulants in vivo.
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The activation of cAMP-dependent protein kinase regulates the physiological activity of AMPA-type glutamate receptors. In this study, phosphorylation of the AMPA receptor subunit GluR1 at Ser(845) was increased in neostriatal slices by activation of D1-type dopamine receptors and by inhibitors of protein phosphatase 1/protein phosphatase 2A. In contrast, Ser(831), a residue which, when phosphorylated by protein kinase C or calcium/calmodulin-dependent kinase II, increases AMPA receptor channel conductance, was unaffected by either D1 or D2 receptor agonists in neostriatal slices. The phosphorylation of Ser(845), but not Ser(831), was strongly increased in neostriatum in vivo in response to the psychostimulants cocaine and methamphetamine. The effects of dopamine and psychostimulants on the phosphorylation of GluR1 were attenuated in dopamine and cAMP-regulated phosphoprotein M(r) 32 kDa (DARPP-32) knock-out mice. These results identify DARPP-32 and AMPA-type glutamate receptors as likely essential cellular effectors for psychostimulant actions. (+info)
Adult learning deficits after neonatal exposure to D-methamphetamine: selective effects on spatial navigation and memory.
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The effects of neonatal d-methamphetamine (MA) treatment on cued and spatial learning and memory were investigated. MA was administered to neonatal rats on postnatal days 11-20. All groups received four subcutaneous injections per day. Group MA40-4 received 40 mg. kg(-1). d(-1) of MA in four divided doses (10 mg/kg per injection). Group MA40-2 received 40 mg. kg(-1). d(-1) of MA in two divided (20 mg/kg/injection) and saline for the other two injections per day. Controls received saline for four injections per day. As adults, both MA groups showed no differences in swimming ability in a straight swimming channel. The MA40-4 group showed no differences in cued learning, but was impaired in hidden platform learning in the Morris water maze on acquisition. They also showed reduced memory performance on probe trials. Similar trends were seen on reversal learning and reversal probe trials. Reduced platform-size learning trials caused spatial learning impairments to re-emerge in the MA40-4 group. The MA40-2 group showed no differences in straight channel swimming, but was slower at finding the visible platform during cued learning. They were also impaired during acquisition and memory trials in the Morris hidden platform maze. They showed a similar trend on reversal learning and memory trials, but were not different during reduced platform-size learning trials. When the MA40-2 group's performance on hidden platform learning and memory trials was adjusted for cued trial performance, the spatial learning deficits remained. Deficits of spatial learning and memory are a selective effect of neonatal methamphetamine treatment irrespective of other learning and performance variables. (+info)
Determination of enantiomeric metabolites of l-deprenyl, d-methamphetamine, and racemic methamphetamine in urine by capillary electrophoresis: comparison of deprenyl use and methamphetamine use.
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The enantiomeric analysis of urine collected from rats administered l-deprenyl, d-methamphetamine (MA), or dl-MA and from healthy male volunteers who ingested l-deprenyl by capillary electrophoresis (CE) using carboxy methylated-beta-cyclodextrin (CMCD) as a chiral selector was investigated to compare the metabolic pattern of l-deprenyl with the metabolism of d- or dl-MA. Urine from illegal drug abusers was also analyzed for the comparison of therapeutic drug (l-deprenyl) use with illicit drug (d-MA) use. MA enantiomers (l-, d-), amphetamine (AM) enantiomers (l-, d-), l-deprenyl, and desmethylselegiline (DMS) enantiomers (l-, d-) were simultaneously separated and detected with clear resolution. L-deprenyl and its metabolites, l-MA, l-AM, and l-DMS, were detected in rat urine sample collected up to 24 h after oral administration of l-deprenyl (10 mg/kg), and the urinary l-AM/l-MA ratio was 2.45 +/- 0.55. This AM/MA ratio was significantly higher than the ratios obtained from rats administered with d-MA (5 mg/kg) and dl-MA (10 mg/kg). The d-AM/d-MA ratio was 0.98 +/- 0.25 for the d-MA treatment, and the d-AM/d-MA and l-AM/l-MA ratios were 0.72 +/- 0.24 and 0.71 +/- 0.21, respectively, for the dl-MA treatment. Analysis of human urine revealed that, unlike in rat urine, the MA content was much greater than the AM content, resulting in the AM/MA ratios being far lower in cases of healthy adult men treated with l-deprenyl (10 mg) and MA abusers. The AM/MA ratio from l-deprenyl users (0.33 +/- 0.03) was significantly higher than the ratio from MA abusers (0.20 +/- 0.12). Results indicate that although metabolic patterns of the drugs in rat and humans may be different, the AM/MA ratio from l-deprenyl use is significantly higher than the ratio from MA use in both rat and human urine. This ratio, however, cannot give conclusive proof of deprenyl or MA use in humans. The simultaneous chiral separation for all the metabolites of l-deprenyl and MA by CE analysis used in this study could provide rapid and simple discrimination between therapeutic drug use and illegal drug abuse. (+info)
Simple and rapid determination of amphetamine, methamphetamine, and their methylenedioxy derivatives in urine by automated in-tube solid-phase microextraction coupled with liquid chromatography-electrospray ionization mass spectrometry.
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A simple and rapid method for the determination of amphetamine, methamphetamine, and their 3,4-methylenedioxy derivatives in urine samples was developed using automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). In-tube SPME is an extraction technique for organic compounds in aqueous samples in which analytes are extracted from the sample directly into an open tubular capillary by repeated draw/eject cycles of sample solution. LC-MS analyses of stimulants were initially performed by liquid injection onto an LC column to determine spectra. Five stimulants tested in this study gave very simple ESI mass spectra, and strong signals corresponding to [M+H]+ were observed for all stimulants. The stimulants were well separated with a Supelcosil LC-CN column using acetonitrile/50mM ammonium acetate (15:85) as a mobile phase. In order to optimize the extraction of stimulants, several in-tube SPME parameters were examined. The optimum extraction conditions were 15 draw/eject cycles of 35 microL of sample in 50mM Tris-HCI (pH 8.5) at a flow rate of 100 microL/min using an Omegawax 250 capillary column. The stimulants extracted by the capillary were easily desorbed by mobile phase flow, and carryover of stimulants was not observed. Using in-tube SPME-LC-ESI-MS with selected ion monitoring, the calibration curves of stimulants were linear in the range from 2 to 100 ng/mL with correlation coefficients above 0.9985 (n = 18) and detection limits (S/N = 3) of 0.38-0.82 ng/mL. This method was successfully applied to the analysis of human urine samples without interference peaks. The recoveries of stimulants spiked into urine samples were above 81%. (+info)
Chemical derivatization and the selection of deuterated internal standard for quantitative determination--methamphetamine example.
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Use of an isotopic analogue of the analyte as the internal standard in a quantitative gas chromatography-mass spectrometry targeted-compound-analysis protocol is often hindered by the availability of an adequate number (typically three for the drug/metabolite and two for the isotopic analogue) of sufficiently high mass ions that can be attributed to each member of the pair and are sufficiently free of interference by the contribution from the other component of the pair, a phenomenon termed "cross-contribution". Methamphetamine (MA) is selected as the exemplar compound to examine the effectiveness in using different chemical derivatization routes to produce derivatized analyte-isotopic analogue pairs that can generate more favorable mass spectrometric data to meet this analytical requirement. Trimethylsilyl-, trichloroacetyl-, and pentafluoropropionyl-derivatization and MA-d5, MA-d8, and MA-d9 are studied. Data resulting from this study indicate that the number of ion pairs suitable for quantitation and the degree of cross-contribution of these ions vary significantly. These data empirically demonstrate that derivatization methods play a significant role in deciding which deuterated analogue of the analyte provides the most suitable ion pairs that cause the least cross-contribution. The most suitable internal standard varies with the derivatization route adapted for an analytical protocol. (+info)