Article Figures & Data
Figures
- Fig. 1.
VMAT2: [3H]DA uptake in subcellular fractions: synaptosomal (A), membrane-associated (B), and vesicular (C). Taar1 WT and KO mice received four intraperitoneal injections of saline or MA (5 mg/kg), 2 hours apart, and were euthanized 24 hours following the final injection. Striatal tissue from four to five mice per genotype per treatment group was pooled and values normalized to the amount of protein in each sample. Experiments were conducted as described in the text. Data represent means ± S.E.M. of five to seven independent experiments, expressed as percentage of the specific uptake in Taar1 WT control (saline) pooled tissue in each experiment. Mean ± S.E.M. of Taar1 WT control group: synaptosomal, 13.4 ± 3.1 pmol/mg protein; membrane-associated, 13.9 ± 3.5 pmol/mg protein; vesicular, 72.9 ± 21.6 pmol/mg. *P < 0.0001 compared with saline-treated controls; +P < 0.05 between genotypes; #P < 0.0001 for the main effect of treatment; †P < 0.01 for the main effect of genotype.
- Fig. 2.
[3H]DHTB saturation binding in subcellular fractions: synaptosomal (A), membrane-associated (B), and vesicular (C). Taar1 WT and KO mice received four intraperitoneal injections of saline or MA (5 mg/kg), 2 hours apart, and were euthanized 24 hours following the final injection. Striatal tissue from 12 to 15 mice per genotype per treatment group was pooled and values normalized to the amount of protein in each sample. Experiments were conducted as described in the text. Shown are representative saturation curves from each fraction. Inset: Scatchard transformation of [3H]DHTB saturation binding data.
- Fig. 3.
DAT: MA-induced changes in [3H]DA uptake in synaptosomes. Striatal synaptosomes from untreated Taar1 WT and KO mice were preincubated with MA (1 nM–10 μM) for 10 minutes before [3H]DA (8 nM) was added and incubated for 10 minutes. Values were normalized to the amount of protein in each sample. Data represent mean ± S.E.M. for each genotype from three independent experiments with two mice of each genotype per experiment, expressed as percentage of the Taar1 WT control (saline) group-specific uptake in each experiment. Mean ± S.E.M. of Taar1 WT control group: 39.9 ± 5.2 pmol/mg protein.
- Fig. 4.
MA-induced [125I]RTI-55 saturation binding in synaptosomes. Taar1 WT and KO mice received four intraperitoneal injections of saline or MA (5 mg/kg), 2 hours apart, and were euthanized 24 hours following the final injection. Values were normalized to the amount of protein in each sample. Experiments were conducted as described in text. Shown is a representative saturation curve. Inset: Scatchard transformation of [125I]RTI-55 binding data.
- Fig. 5.
Effects of repeated saline or MA injections on core body temperature. Taar1 WT and KO mice received four intraperitoneal injections (indicated by arrows) of saline (A) or MA (B; 5 mg/kg), 2 hours apart. Body temperature was measured every 15 minutes via telemetry over 8 hours in an ambient temperature environment of 23 ± 1°C. Data represent temperature for each genotype and treatment group (mean ± S.E.M.) at specified time points, n = 11–12 mice per genotype per treatment group. Time points selected for detailed analysis were 30 minutes after each injection. *P < 0.01; **P < 0.0001 between genotypes.
Tables
- TABLE 1
[3H]DHTB binding in subcellular fractions
Data represent mean ± S.E.M. for each treatment group from three independent experiments, using pooled tissue from 12–15 mice per genotype per treatment group.
Fraction Treatment Genotype Bmax ± S.E.M. Kd ± S.E.M. pmol/mg protein nM Synaptosomal Saline Taar1 WT 1.4 ± 0.2 2.3 ± 0.5 Taar1 KO 1.5 ± 0.2 2.4 ± 0.4 MA Taar1 WT 1.3 ± 0.2 2.3 ± 0.4 Taar1 KO 1.3 ± 0.2 2.3 ± 0.2 Membrane-associated Saline Taar1 WT 1.9 ± 0.3 2.3 ± 0.5 Taar1 KO 2.0 ± 0.5 2.3 ± 0.7 MA Taar1 WT 1.9 ± 0.3 2.3 ± 0.6 Taar1 KO 1.5 ± 0.3 2.4 ± 0.5 Vesicular Saline Taar1 WT 1.0 ± 0.2 2.2 ± 0.3 Taar1 KO 1.2 ± 0.1 3.4 ± 1.0 MA Taar1 WT 0.8 ± 0.2* 1.2 ± 0.1* Taar1 KO 0.6 ± 0.1* 1.1 ± 0.1* * P < 0.05 compared with saline treatment (main effect).
- TABLE 2
[125I]RTI-55 specific binding in subcellular fractions
Data represent mean ± S.E.M. for each subcellular fraction from three independent experiments, using pooled tissue from six to eight untreated Taar1 HET mice with one to two independently pooled preparations of tissue per experiment.
Fraction [125I]RTI-55 Binding ± S.E.M. pmol/mg protein Synaptosomal 3.9 ± 0.4* Membrane-associated 6.2 ± 0.5* Vesicular 0.6 ± 0.1* * P < 0.01; fractions significantly differed from each other.
- TABLE 3
[125I]RTI-55 binding in synaptosomes
Data represent mean ± S.E.M. for each genotype and treatment group from four independent experiments, n = 4 mice per genotype per treatment group.
Treatment Genotype Bmax S.E.M. Kd ± S.E.M. pmol/mg protein nM Saline Taar1 WT 10.3 ± 0.8 3.7 ± 0.2 Taar1 KO 11.7 ± 1.3 4.3 ± 0.4 MA Taar1 WT 8.5 ± 0.4* 3.6 ± 0.4 Taar1 KO 7.6 ± 1.0* 3.8 ± 0.7 * P < 0.01 compared with saline treatment (main effect).
- TABLE 4
MA-induced change in body temperature relative to saline-treated mice
Data represent change (ΔT) in temperature (mean ± S.E.M.) for each treatment group from saline-treated mice at the specified time point; n = 11–12 mice per genotype per treatment group. Taar1 WT saline-treated group body temperature at each timepoint (mean ± S.E.M.): 1st inj, 37.9 ± 0.1°C; 2nd inj, 37.7 ± 0.1°C; 3rd inj, 37.3 ± 0.1°C; 4th inj, 37.1 ± 0.1°C. Taar1 KO saline-treated group body temperature at each timepoint: 1st inj, 37.7 ± 0.2°C; 2nd inj, 37.6 ± 0.1°C; 3rd inj, 37.2 ± 0.1°C; 4th inj, 36.7 ± 0.1°C.
Genotype MA Dose (mg/kg) 30 Min Post-Injection (ΔT °C ± S.E.M.) 1st Inj 2nd Inj 3rd Inj 4th Inj Taar1 WT 5 −2.0 ± 0.2* −0.7 ± 0.3** −0.1 ± 0.2 −0.3 ± 0.2 Taar1 KO 5 −0.2 ± 0.2 0.5 ± 0.1** 0.3 ± 0.2 −0.2 ± 0.2
