Strain differences in basal and cocaine-evoked dopamine dynamics in mouse striatum.
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In vivo microdialysis was used to characterize basal dopamine (DA) dynamics and cocaine-evoked DA levels in the striatum of 129/Sv-ter, C57BL/6J, DBA/2J, and Swiss-Webster mice. Basal dialysate levels of DA did not differ in the four strains tested. Similarly, the no net flux method of quantitative microdialysis revealed no difference in extracellular levels between strains. However, the in vivo extraction fraction of DA was significantly less in 129/Sv-ter (53%) mice compared with C57BL/6J (68%), DBA/2J (69%), and Swiss-Webster (67%) mice, indicating a lower rate of basal DA uptake in the 129/Sv-ter strain. Perfusion of K(+) (60 and 100 mM) through the microdialysis probe significantly increased dialysate DA levels and there was no difference between strains in the magnitude of this effect. The acute administration of cocaine (5-20 mg/kg i.p.) increased DA levels in the four strains tested. Cocaine-evoked DA levels (in nanomoles) were significantly greater in 129/Sv-ter compared with C57BL/6J, DBA/2J, or Swiss-Webster mice after administration of either 5, 10, or 20 mg/kg cocaine. However, the percentage increase in DA did not differ across strains. These data demonstrate that there are strain-related differences in basal DA dynamics in the striatum of the mouse. Basal DA uptake was reduced in striatum of 129/Sv-ter mice compared with C57BL/6J, DBA/2J, or Swiss-Webster mice. In addition, the response of DA levels to cocaine may be enhanced in 129/Sv-ter compared with C57BL/6J, DBA/2J, or Swiss-Webster mice. (+info)
Cocaine-induced seizure thresholds: quantitative trait loci detection and mapping in two populations derived from the C57BL/6 and DBA/2 mouse strains.
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Seizures are a well known consequence of human cocaine abuse, and in rodent models, sensitivity to cocaine seizures has been shown to be strongly influenced by genotype. For example, several studies have reported significant differences between the C57BL/6 (B6) and DBA/2 (D2) inbred mouse strains in their sensitivity to cocaine-induced seizures. This prompted our use of the BXD recombinant inbred (RI) strain set and an F(2) population derived from the B6 and D2 progenitor strains for further genetic analyses and for gene mapping efforts in this study. Cocaine was infused into the lateral tail vein, and the doses needed to induce a running bouncing clonic seizure and a tonic hindlimb extensor seizure were recorded for each mouse. In the BXD RI set, a genome-wide search was carried out for QTLs (quantitative trait loci), which are sites on a chromosome containing genes that influence seizure susceptibility. An F(2) population (B6D2F2, n = 408) was subsequently used as a second, confirmation step. Based on both RI and F(2) results, three QTLs emerged as significant (P <.00005): one for clonic seizures on chromosome 9 (distal), and two for tonic seizures on chromosomes 14 (proximal to mid) and 15 (distal). Two additional QTLs emerged as suggestive (P <.0015), both associated with clonic seizures on chromosomes 9 (proximal) and 15 (distal). Both QTLs on chromosome 9 were sex-specific, with much larger effects on the phenotype seen in females than in males. (+info)
In vitro studies of striatal vesicles containing the vesicular monoamine transporter (VMAT2): rat versus mouse differences in sequestration of 1-methyl-4-phenylpyridinium.
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Significant differences exist in the sensitivity of mice and rats to the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that cannot be explained by differences in exposure to or uptake of 1-methyl-4-phenylpyridinium (MPP(+)) into dopamine (DA) neurons. MPP(+) is also a substrate for the brain vesicular monoamine transporter (VMAT2), and sequestration into synaptic vesicles may be one mechanism of protection against MPP(+) toxicity. A greater sequestration of MPP(+) into vesicles of DA neurons in rats versus mice could explain the lower vulnerability of DA neurons in the rat to MPP(+) toxicity. To test this hypothesis, the kinetics of uptake for [(3)H]MPP(+) and [(3)H]DA as well as [(3)H]dihydrotetrabenazine binding to VMAT2 were compared in vesicles isolated from the striata of rats and mice. The K(m) value of [(3)H]MPP(+) transport was similar in the two species. In contrast, the maximal transport rate (V(max)) was 2-fold greater in vesicles from rats than in those from mice. Likewise, the K(m) value for [(3)H]DA transport was similar in both preparations, but the V(max) value was 2-fold greater in rat than in mouse vesicles. The B(max) value for [(3)H]dihydrotetrabenazine binding was also 2-fold greater in striatal vesicles from rats than in those from mice. Electron micrographs demonstrated that vesicles isolated from rats and mice were approximately the same size. Based on these observations, we propose that striatal vesicles from rats have more VMAT2 than vesicles from mice and that this species difference in VMAT2 density may help explain the reduced vulnerability of rat DA neurons to MPP(+) neurotoxicity. (+info)
Cocaine potentiates ethanol-induced excitation of dopaminergic reward neurons in the ventral tegmental area.
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The coabuse of cocaine and ethanol is one of the most frequently used substance abuse combinations in the United States. The dopamine (DA) neurons in the ventral tegmental area (VTA) are important in the rewarding mechanism of these two substances. Cocaine is known to block the reuptake of DA and serotonin (5-HT). At concentrations below 1 microM, cocaine preferentially blocks the reuptake of 5-HT compared with DA. We have previously shown that ethanol increases the firing rate of DA neurons in the VTA, and that this excitation is enhanced by 5-HT. Extracellular single-unit recordings were made from VTA dopaminergic neurons in coronal brain slices from young adult Fischer 344 rats. Cocaine (1-10 microM) reduced the spontaneous firing rate in VTA dopaminergic neurons in a concentration-related manner. A lower concentration of cocaine (500 nM), which is a concentration that is pharmacologically relevant in addicts, produced only a very small decrease in the firing rate of VTA neurons but potentiated ethanol excitation of these neurons. Higher concentrations of cocaine (1 microM) did not enhance ethanol excitation. Ethanol-induced excitation was potentiated by the higher concentrations of cocaine (1 and 2 microM) in the presence of the D(2) receptor antagonist sulpiride (1 microM). Furthermore, cocaine potentiation of ethanol-induced excitation was reversed by ketanserin (2 microM), a 5-HT(2) antagonist. The enhanced ethanol excitation of VTA dopaminergic neurons caused by cocaine may partially explain the high incidence of the coabuse of these two substances. (+info)
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)
Characterization of a tropane radioligand, [(3)H]2beta-propanoyl-3beta-(4-tolyl) tropane ([(3)H]PTT), for dopamine transport sites in rat brain.
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PTT (2beta-propanoyl-3beta-[4-tolyl] tropane) is a tropane analog relatively selective for dopamine transporters in binding and uptake assays in vitro, with long-acting psychostimulant properties in vivo. To explore its utility in binding to dopamine transporters, [(3)H]PTT was synthesized and assayed for binding in rat striatal membranes and by in vitro autoradiography. In membranes, binding of [(3)H]PTT was saturable to a single class of binding sites with a K(D) value of 3 nM. The pharmacology of [(3)H]PTT binding in striatal membranes was consistent with that of a ligand selective for dopamine transporters, with dopamine-selective compounds being significantly more potent in displacing [(3)H]PTT binding than those for 5-HT or norepinephrine transporters. Although the ability of various transporter inhibitors to displace both [(125)I]RTI-55 and [(3)H]PTT binding correlated significantly with each other, there was a better correlation of inhibitor potencies versus [(3)H]PTT binding and dopamine uptake than versus [(125)I]RTI-55 binding and dopamine uptake. The differences in correlations were most noticeable for compounds relatively selective at the 5-hydroxytryptamine (serotonin) transporter. The autoradiographic distribution of [(3)H]PTT binding in coronal sections was consistent with the known distribution of the dopamine transporter, with high levels of binding evident in caudate nucleus, nucleus accumbens, and olfactory tubercle. Moderate densities of [(3)H]PTT binding were also observed in substantia nigra pars compacta, and ventral tegmental area, as well as in the anterior cingulate cortex and portions of the hypothalamus. In addition, nonspecific binding was less than 5% of total binding. Thus, [(3)H]PTT provides an accurate and convenient marker for the dopamine transporter. (+info)
Analysis of four dopaminergic tracers kinetics using two different tissue input function methods.
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The integrity of the dopaminergic system can be studied using positron emission tomography. The presynaptic tracers [11C]-methylphenidate and [11C]dihydrotetrabenazine (DTBZ) are used to investigate the dopamine transporter availability, the dopamine vesicular transporter integrity; the postsynaptic tracers [11C]-raclopride and [11C]-Schering 23990 (SCH) are used to probe the D2 and D1 receptors. These are reversible tracers, where the binding potential (BP) = Bmax/Kd often is used to quantify the amount of their specific binding to the sites of interest. The simplified tissue input methods to calculate BP are attractive, since they do not require a blood input function. The suitability and performance of two such methods were evaluated: the Logan graphical tissue method, and the Lammertsma reference tissue method (RTM). The BP estimates obtained with the two methods were nearly identical in most cases, with similar reliability and reproducibility indicating that all four tracers satisfy the assumptions required by each method. The correlations among the fitted parameters obtained from the RTM were estimated and were found not to introduce noticeable bias in the RTM BP and R1 estimates. R1 showed low intersubject and intrasubject variability. The k2 estimate showed good reliability for SCH with cerebellar input function and DTBZ with occipital input function. (+info)
Importance of valine at position 152 for the substrate transport and 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane binding of dopamine transporter.
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Human and bovine dopamine transporters (DAT) demonstrate discrete functional differences in dopamine (DA), 1-methyl-4-phenylpyridium (MPP(+)) transport, and cocaine analog binding. In a previous study, the functional analyses on the chimeras of human and bovine DAT have revealed that the region from residues 133 through 186 (encompassing the third transmembrane domain) is responsible for the substrate transport and cocaine analog binding. The present study has been carried out to determine the specific amino acid(s) conferring DAT functions by interchanging the amino acid residues in the corresponding region between human and bovine DAT. As described previously, the DA, MPP(+) transport, and 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) binding almost disappeared in chimera hb3 in which the region from residues 133 through 186 of bovine DAT was substituted into human DAT. Replacement of isoleucine, residue 152 of chimera hb3 (bovine DAT sequence), with valine, the human DAT residue at the identical position, remarkably restored the substrate transport and CFT binding to 76% to 98% of the human DAT values. Similarly, substitution of isoleucine for valine at position 152 in the human DAT reduced the substrate transport and CFT binding by 57% to 97%. Among other amino acids tested at position 152 of the chimera hb3, only alanine resulted in small but significant increases in the DAT functions ranging from 16 to 34%. Thus, valine at position 152 plays a crucial role for molecular mechanisms underlying the interactions of DA, MPP(+), and CFT with human DAT. (+info)