Figure 3
(a) [3H]dopamine uptake kinetics in striatal synaptosomes of wild type (WT) and knockout (KO) animals: striatal synaptosomes were incubated with 0.1 μM [3H]dopamine and increasing concentrations of unlabeled dopamine (0–3 μM) for 5 min at 37 °C. Non-specific uptake was determined in the presence of 1 μM GBR12909. Kd and Bmax values are not significantly different (Km: WT: 88.3±30.7 nM, αCaMKII-KO: 111.2±47.1 nM; Vmax: WT: 4.66±0.32 pmol/mg protein/min, αCaMKII-KO: 4.87±0.45 pmol/mg protein/min; WT n=6, KO n=5; p>0.05). (b) Fast-scan cyclic voltammetry (FSCV) was used to measure dopamine release from striatal brain slices. Representative traces in control and KO animal exemplify the higher peak height in mutants compared with control. (c) Average of stimulated dopamine release in dorsal striatum of control and KO animals show an increased evoked dopamine release in KO animals (n=6; *p<0.05, unpaired Student’s t-test). (d) Densitometric analysis of VMAT2 protein levels in the striatum of WT and KO mice: bands were normalized to tubulin; n=9–10; Student’s t-test, p>0.05 (e) Uptake of [3H]dopamine in striatal synaptic vesicles: vesicles were incubated with 40 nM [3H]dopamine for 10 min at 30 °C. Non-specific uptake was determined in the presence of 10 μM reserpine; n=3; p>0.05 (f) Dopamine D1 receptor levels as assessed by [3H]SCH23390 binding: WT Bmax=326.6±33.2 fmol/mg protein, KO Bmax=287.1±32.9 fmol/mg protein, Kd: WT Kd=1.55±0.76 nM, KO Kd=1.50±0.81 nM; WT n=4, KO n=5). (g) Dopamine D2 receptor levels as assessed by [3H]raclopride: WT Bmax=280.2±30.0 fmol/mg protein, n=6; KO Bmax=229.0±19.7 fmol/mg protein, n=5; WT Kd=7.18±2.15 nM, KO Kd=2.55±0.84 nM. Kd and Bmax values are not significantly different; Student’s t-test, p>0.05. (h) Densitometric analysis of PSD-95 protein levels in the striatum of WT and KO mice: bands were normalized to tubulin; n=7; Student’s t-test, p>0.05.
