Journal: Frontiers in Cellular Neuroscience
Article Title: Striatal Dopamine Transporter Function Is Facilitated by Converging Biology of α-Synuclein and Cholesterol
doi: 10.3389/fncel.2021.658244
Figure Lengend Snippet: Dopamine transporters (DAT) function is potentiated by α-synuclein. (A) Mean [DA] o ± SEM ( shaded ) evoked by single pulses ( arrow ) vs. time in caudate putamen (CPu) of Snca -null (dashed line) and SNCA -OVX (solid line) mice in drug-free conditions ( left ) or DHβE ( right ). (B) Summary of peak evoked [DA] o . *** P < 0.001, two-way ANOVA main effect of genotype: F (1,90) = 14.54, P = 0.0003; drug x genotype interaction: F (1,90) = 3.19, P = 0.078. (C,D) One-phase exponential decay curve fits for falling phases of the mean of concentration-matched [DA] o transients evoked by a single pulse (C) or 20p 20 Hz in the presence of DHBE (1 μM), 4-aminopyridine (100 μM), L-741,626 (1 μM) (D) , for Snca -null and SNCA -OVX mice. Insets , transients offset to allow for concentration-matching (arrow) . Comparison of k , Snca -null vs. SNCA -OVX: 1p, 2.39 vs. 2.92, F (1,825) = 64.5, P < 0.0001; 20p, 1.29 vs. 1.73, F (1,842) = 53.01, P < 0.0001. (E) Summary of k (s −1 ) calculated for all transients, 2.34 vs. 3.13, t (123) = 3.35, ** P = 0.0011. (F) Maximum decay rates seen for each transient vs. [DA] o at that rate for Snca -null (unfilled) and SNCA -OVX (filled). Unconstrained Michelis–Menten curve-fits for null ( dashed ) and SNCA -OVX (solid). V max and K m are indicated by horizontal and vertical lines. V max = 7.20 vs. 11.84 μM/s, K m = 1.82 vs. 3.97 μM, comparison of fits: F (2,181) = 15.75, P < 0.001, R 2 = 0.52 and 0.80. (G) Mean [DA] o (μM) ± SEM vs. time evoked by single pulses ( arrow ) in CPu, before cocaine (control, black line) and in the presence of cocaine (blue, 5 μM). (H) Summary of effects of DAT inhibitors cocaine, GBR 12935 and nomifensine on 1p-evoked [DA] o in Snca -null and SNCA -OVX, two-way ANOVA: effect of drug, F (2,28) = 18.63, P < 0.0001; effect of genotype, F (1,28) = 18.67, P = 0.0002; genotype x cocaine interaction, F (2,28) = 0.91, P = 0.41. (I) Paired-pulse ratios (PPR) for [DA] o vs. inter-pulse interval (IPI) in control conditions (DHβE; black ), and with cocaine ( blue ) in Snca -null (unfilled) and SNCA -OVX (filled). Two-way ANOVA: condition x IPI interaction, F (12,100) = 137.9, P < 0.0001, Sidak’s post-test *** P < 0.001. (J) Peak [DA] o (normalized to condition 1p) ± SEM vs. frequency for 4p trains (5–100 Hz) in control (in the presence of DHβE; black ) and cocaine ( blue ) in Snca -null (unfilled) and SNCA -OVX (filled). Two-way ANOVA: condition x frequency interaction, F (12,40) = 19.56, P < 0.0001, Sidak’s posttest: *** P < 0.001. Insets, normalized effect of cocaine on 1p-evoked release for these datasets.
Article Snippet: We used exponential decay curve fits and Michaelis-Menten models to fit the falling phases of evoked DA transients using GraphPad Prism 6.0.
Techniques: Concentration Assay, Comparison, Control
GraphPad Software Inc is a verified supplier 
![Dopamine transporters (DAT) function is potentiated by α-synuclein. (A) Mean [DA] o ± SEM ( shaded ) evoked by single pulses ( arrow ) vs. time in caudate putamen (CPu) of Snca -null (dashed line) and SNCA -OVX (solid line) mice in drug-free conditions ( left ) or DHβE ( right ). (B) Summary of peak evoked [DA] o . *** P < 0.001, two-way ANOVA main effect of genotype: F (1,90) = 14.54, P = 0.0003; drug x genotype interaction: F (1,90) = 3.19, P = 0.078. (C,D) One-phase <t>exponential</t> decay curve fits for falling phases of the mean of concentration-matched [DA] o transients evoked by a single pulse (C) or 20p 20 Hz in the presence of DHBE (1 μM), 4-aminopyridine (100 μM), L-741,626 (1 μM) (D) , for Snca -null and SNCA -OVX mice. Insets , transients offset to allow for concentration-matching (arrow) . Comparison of k , Snca -null vs. SNCA -OVX: 1p, 2.39 vs. 2.92, F (1,825) = 64.5, P < 0.0001; 20p, 1.29 vs. 1.73, F (1,842) = 53.01, P < 0.0001. (E) Summary of k (s −1 ) calculated for all transients, 2.34 vs. 3.13, t (123) = 3.35, ** P = 0.0011. (F) Maximum decay rates seen for each transient vs. [DA] o at that rate for Snca -null (unfilled) and SNCA -OVX (filled). Unconstrained Michelis–Menten curve-fits for null ( dashed ) and SNCA -OVX (solid). V max and K m are indicated by horizontal and vertical lines. V max = 7.20 vs. 11.84 μM/s, K m = 1.82 vs. 3.97 μM, comparison of fits: F (2,181) = 15.75, P < 0.001, R 2 = 0.52 and 0.80. (G) Mean [DA] o (μM) ± SEM vs. time evoked by single pulses ( arrow ) in CPu, before cocaine (control, black line) and in the presence of cocaine (blue, 5 μM). (H) Summary of effects of DAT inhibitors cocaine, GBR 12935 and nomifensine on 1p-evoked [DA] o in Snca -null and SNCA -OVX, two-way ANOVA: effect of drug, F (2,28) = 18.63, P < 0.0001; effect of genotype, F (1,28) = 18.67, P = 0.0002; genotype x cocaine interaction, F (2,28) = 0.91, P = 0.41. (I) Paired-pulse ratios (PPR) for [DA] o vs. inter-pulse interval (IPI) in control conditions (DHβE; black ), and with cocaine ( blue ) in Snca -null (unfilled) and SNCA -OVX (filled). Two-way ANOVA: condition x IPI interaction, F (12,100) = 137.9, P < 0.0001, Sidak’s post-test *** P < 0.001. (J) Peak [DA] o (normalized to condition 1p) ± SEM vs. frequency for 4p trains (5–100 Hz) in control (in the presence of DHβE; black ) and cocaine ( blue ) in Snca -null (unfilled) and SNCA -OVX (filled). Two-way ANOVA: condition x frequency interaction, F (12,40) = 19.56, P < 0.0001, Sidak’s posttest: *** P < 0.001. Insets, normalized effect of cocaine on 1p-evoked release for these datasets.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1845/pmc08081845/pmc08081845__fncel-15-658244-g0001.jpg)
![Applied cholesterol potentiates DAT function. (A) Mean [DA] o ± SEM vs. time evoked by single pulses ( arrow ) in CPu from Snca -null mice incubated in vehicle or ws-cholesterol. **** P < 0.0001. (B) One-phase exponential decay curve fits for falling phases of mean [DA] o transients concentration-matched. k (s −1 ) 2.54 (vehicle) vs. 3.14 (cholesterol), half-lives ( t (1/2) ) are indicated by for vehicle (fine line) and cholesterol (thick dotted), F (1,617) = 46.4, *** P < 0.0001. (C) Mean [DA] o (normalized to control conditions) ± SEM vs. time evoked by single pulses ( arrow ) in CPu of Snca -null mice, in control ( black ) and in the presence of cocaine ( blue ) from slices incubated in vehicle or cholesterol. **** P < 0.0001. (D) Summary of effects of cocaine on mean peak [DA] o ± SEM, normalized to pre-cocaine condition. Two-way ANOVA: effect of cholesterol, F (1,8) = 14.9, *** P = 0.005; effect of genotype, F (1,8) = 15.8, P = 0.004; Genotype x cocaine interaction, F (1,8) = 0.15, P = 0.70. (E) Secondary ion mass spectrometry (SIMS) analyses of Snca -null (top) and SNCA -OVX (bottom) striatum groups obtained from the striatum region of brain tissue in positive ion mode. Cholesterol (green arrow) and galactosylceramide ( galcer ; purple arrow) signal intensity (normalized to the number of selected pixels for the spectrum). (F) Statistical comparison of striatal lipid species cholesterol and galcer (32:1). In SNCA−/− (unfilled bars) and SCNA -OVX (filled bars) Mean ± SEM (normalized to the total ion count). 2-sample T -test, n = 12, *** P < 0.001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1845/pmc08081845/pmc08081845__fncel-15-658244-g0003.jpg)