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.2013 Oct 14;8(10):e75975.
doi: 10.1371/journal.pone.0075975. eCollection 2013.

Methamphetamine increases locomotion and dopamine transporter activity in dopamine d5 receptor-deficient mice

Affiliations

Methamphetamine increases locomotion and dopamine transporter activity in dopamine d5 receptor-deficient mice

Seiji Hayashizaki et al. PLoS One..

Abstract

Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behavioral response to methamphetamine. Interestingly, D5 dopamine receptor-deficient mice displayed increased ambulation in response to methamphetamine. Furthermore, dopamine transporter threonine phosphorylation levels, which regulate amphetamine-induced dopamine release, were elevated in D5 dopamine receptor-deficient mice. The increase in methamphetamine-induced locomotor activity was eliminated by pretreatment with the dopamine transporter blocker GBR12909. Taken together, these results suggest that dopamine transporter activity and threonine phosphorylation levels are regulated by D5 dopamine receptors.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of D5R-KO mice.
(a) Design of the D5R gene targeting vector. Upper diagram: restriction enzyme map for the WT D5R gene locus. The black part of the box corresponds to the D5R gene coding region and the white part of the box represents the noncoding region. Middle diagram: the D5R gene targeting vector. Lower diagram: the D5R gene locus in the D5R-KO mice. Bottom diagram: Probes used for recombinant ES cell screening are indicated. (b) Genomic Southern blotting with a 3′ region probe. Genomic DNA was collected from WT (+/+), heterogeneous (+/−), and homogenous (−/−) D5R mice and subjected to electrophoresis and Southern blotting. The bands corresponding to wild-type and mutant DNA are indicated. (c) mRNA was collected from WT (+/+), heterogeneous (+/−), and homogenous (−/−) animals and subjected to electrophoresis and Northern blotting with a D5R cDNA probe. D5R mRNA was absent from the homogenous (−/−) D5R-KO animals.
Figure 2
Figure 2. Increased METH-induced ambulatory activity in D5R-KO mice.
(a) Open field locomotor activity after challenge with METH (2.5 mg/kg). Locomotion was measured for 60 min following each injection. The data were presented in 10 min time bins. The circles or squares represent the mean and the error bars represent the s.e.m. The pretreatments (arrowhead) were either saline (WT, filled circles,n = 6; and D5R-KO, open circles,n = 6) or GBR12909 (5 mg/kg) (WT, filled squares,n = 6; and D5R-KO, open squares,n = 6). The pretreatments were administered 80 minutes before the METH challenge (arrow). The secondary interaction between blocker pretreatment, genotype, and time course was: F(11,220) = 3.08; andp<0.001. The interactions between blocker and genotype at various time points (20, 30, 40, and 50 min after the METH challenge) were F(1,240) = 5.29, 5.63, 4.04, and 6.01; andp<0.05at all time points. The main effects of genotype (saline pretreatments) at various time points (10, 20, 30, 40, 50, and 60 min after the METH challenge) were F(1,240) = 4.08, 18.07, 20.08, 15.01, 15.58, and 7.56; andp = <0.05,p<0.0001,p<0.0001,p<0.001,p<0.001, andp<0.01, respectively. The main effects of GBR12909 on D5R-KO mice at various time points (10, 20, and 50 min after the METH challenge) were F(1,240) = 5.37, 7.99, and 4.68; andp<0.05×10−2,p<0.01, andp<0.05, respectively. *p<0.05 for main effect of genotype; and #p<0.05 for main effect of blocker. (b) Open field locomotor activity after challenge with cocaine (15 mg/kg). Ambulation of two experimental groups of mice (n = 8 each) after cocaine challenge. Saline injection (arrowhead) was performed 80 minutes before the cocaine challenge (arrow). WT, filled circles; D5R-KO, open circles. The interaction between genotype and challenge was F(1,154) = 1.00 andp = 4.49×10−1.
Figure 3
Figure 3. DAT phosphorylation in D5R-KO mice.
DAT proteins were immunoprecipitated from whole brain lysates and were then immunoblotted with anti-phosphothreonine and anti-DAT antibodies. (a) Upper: Representative Western blot of phospho-threonine signals of immunoprecipitated DAT proteins. Lower: Total DAT protein levels of the same samples. WT, D5R-KO, and DAT-KO genotypes are indicated. (b) The intensities of the phosphothreonine bands were normalized to the intensities of the total DAT protein bands to quantify the phosphothreonine levels. WT mice, black bar; D5R-KO mice, gray bar. Threonine phosphorylation levels were significantly increased in D5R-KO mouse brains relative to WT mouse brains. Paired t-test:t = −2.59 andp<0.05. *p<0.05.
Figure 4
Figure 4. DAT levels in D5R-KO mice.
(a) Dialysate samples were collected at a sampling rate of 2 µl/min for 20 min during a baseline period of 60 min and then for an experimental period of 120 min following the METH challenge (2.5 mg/kg; arrow). The interaction between blocker pretreatment and challenge was F(5,120) = 6.66 andp<0.0001. The simple main effects of blocker pretreatment at 40 and 60 min were F(1,144) = 13.50 and 8.08, andp<0.001 andp<0.001, respectively. WT, filled circles (n = 7); D5R-KO, open circles (n = 7). DA, dopamine. The arrowhead indicates the time point of saline injection. (b) Dialysate samples were collected for 20 min during a baseline period of 60 min. The animals were then injected with GBR12909 (5 mg/kg) and samples were collected for 20 min during a pretreatment period of 80 min. The animals were then challenged with METH (2.5 mg/kg) and samples were collected throughout the 120 min experimental period. The arrowhead indicates the GBR12909 pretreatment and the arrow indicates the METH challenge. (c) Representative microdialysis probe placements. Dashed lines denote the boundaries of the NA and the anterior commissure (ac). (d) Schematic representations of probe placements in the experimental groups for microdialysis of the NA at two different rostrocaudal levels (1.1 mm and 0.8 mm rostral from the bregma). The short lines indicate the probe tracks. Seven WT and seven D5R-KO probe track cases are overlaid on representative sections.
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This work was supported by the Core Research for Evolutional Science and Technology (CREST) program of the Japan Science and Technology (JST) Agency. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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