Fig 1: Enrichment of GAT-1 and GAT-3 expression in the DLS versus NAcC.a, c Representative immunofluorescence signals for GAT-1 (cyan, a) and GAT-3 (green, c) using confocal microscopy in coronal sections across the rostral-caudal limits containing striatum prepared from an individual C57BL/6 J mouse with heatmaps for striatal GAT intensity. Boxes indicate representative locations for GAT intensity measurements in the dorsolateral striatum (DLS) and nucleus accumbens core (NAcC). Scale bars: 1 mm. Note enriched GAT-3 in the medial NAc shell contiguous with the medial septal nucleus and enriched GAT-3 expression in the claustrum. b, d Left, Mean GAT-1 (b) and GAT-3 (d) intensity in DLS and NAcC normalized to total striatum and averaged across rostral-caudal sites for each hemisphere (n = 12 hemispheres/6 mice for each GAT-1 and GAT-3). Right, Representative single plane images of GAT-1 (b) and GAT-3 (d) immunofluorescence from DLS and NAcC; imaging parameters were kept constant across regions. Scale bars: 50 μm. Mann–Whitney U tests (b, d). **p < 0.01. Error bars are ±SEM. Source data are provided as a Source Data file.
Fig 2: Enhanced tonic inhibition of striatal DA release and impaired GAT function in mouse model of early parkinsonism.a, b, e, i Mean peak [DA]o during consecutive recordings evoked by a single (1p) light (a, b, i) or electrical pulse (e) in DLS (a, e, i) or NAcC (b) during applications of antagonists for GABAA (bicuculline, 10 µM) and GABAB receptors (CGP 55845, 4 µM) (a, b, i), or the nonspecific GAT inhibitor NPA (1.5 mM) (e) in slices preincubated (i) or not preincubated (a, b, e) with fluorocitrate (FC, 200 µM, 45–60 min) from Snca−/− (dark blue, GABAR antagonism: n = 7 experiments/5 mice in DLS (a), n = 9 experiments/5 mice in NAcC (b), n = 7 experiments/3 mice for fluorocitrate (i); NPA: n = 5 experiments/4 mice (e) and SNCA+ mice (light blue, GABAR antagonism: n = 8 experiments/5 mice for both DLS and NAcC (a, b), n = 6 experiments/3 mice for fluorocitrate (i); NPA: n = 5 experiments/4 mice (e). Data are normalized to mean of four timepoints prior to drug application (dotted line); mean transients of [DA]o are derived from last four timepoints (grey shaded region) and normalized to predrug baselines. c, d, f, g, j, k Cumulative frequency plots of individual data points (c, f, j) and mean per recording site (d, g, k) from a, b, e, i. h Representative Western blots and mean GAT-1 and GAT-3 protein content of dorsal striatum tissue taken from Snca−/− mice (n = 10 mice) and SNCA+ mice (n = 7 mice). Data normalized to actin and littermate control expression. Two-way repeated-measures ANOVA with Sidak’s multiple comparison tests (a, b, e, i), Komogorov–Smirnov tests (c, f, j), two-tailed Student’s unpaired t-tests (d, g, k) and Mann–Whitney U tests (h). *p < 0.05, **p < 0.01, ***p < 0.001. Error bars are ±SEM. Source data and uncropped blots from h are provided as a Source Data file.
Fig 3: GAT-1 and GAT-3 inhibition attenuates DA release in DLS, but not NAcC.a, b, d, e, g, h Mean peak [DA]o during consecutive recordings evoked by a single electrical pulse (1p) in DLS (a, d, e, g, h) or NAcC (b) in control conditions (black, n = 9 experiments/7 mice for DLS, n = 6 experiments/5 mice for NAcC) or with GAT inhibitor nipecotic acid (NPA, 1.5 mM) (a, blue, n = 9 experiments/5 mice; b, n = 6 experiments/4 mice), combined bath application of the GAT-1 specific inhibitor SKF89976A (20 μM) and the GAT-3 specific inhibitor SNAP5114 (50 μM) (d, green, n = 9 experiments/5 mice), SKF89976A alone (e, orange, n = 6 experiments/4 mice), SNAP5114 alone (g, purple, n = 6 experiments/4 mice), or bath application of SNAP5114 in slices preincubated in SKF89976A (h, red, n = 7 experiments/5 mice; black, SKF89976A preincubated controls, n = 6 experiments/3 mice). c, f, i Mean peak [DA]o evoked by 1p following GAT inhibition (expressed as a % of predrug baseline). j Mean DA content of dorsal striatum incubated in vehicle-treated control conditions (black, n = 19 punches/5 mice) or NPA (1.5 mM) (blue, n = 19 punches/5 mice). k Mean peak [DA]o during consecutive recordings evoked by 1p in DLS during application of NPA (1.5 mM) in the absence (light blue, n = 9 experiments/5 mice) or presence (dark blue, n = 5 experiments/4 mice) of GABAA (picrotoxin, 100 μM) and GABAB (CGP 55845, 4 μM) receptor antagonists. l Left, Mean peak values of [DA]o evoked by 50 Hz electrical pulses in DLS normalized to 1p in the absence (black, control, n = 8 experiments/5 mice) or presence of NPA (1.5 mM) (blue, n = 8 experiments/5 mice). Sigmoidal curve fits (R2 = 0.98). Data are normalized to mean of four timepoints prior to GAT inhibitor application (dotted line); mean transients of [DA]o are derived from last four timepoints (grey shaded region) and normalized to predrug baselines. DHβE (1 μM) present throughout. Two-way repeated-measures ANOVA with Sidak’s multiple comparison tests (a, b, d, e, g, h, k, l), two-tailed Student’s unpaired t-tests (c, i), Mann–Whitney test (j) and one-way ANOVA with Sidak’s multiple comparison tests (f). *p < 0.05, **p < 0.01, ***p < 0.001. Control data in d, e, g are the same as in a. Error bars are ±SEM. Source data are provided as a Source Data file.
Fig 4: dlVP astroglia exhibited increased GAT-3 expression and synaptic co-registration during 15 min of cued sucrose seeking.A Male and female rats were trained to self-administer sucrose during daily 2 h sessions. After extinction training, rats reinstated sucrose seeking during 15-min exposure to sucrose-associated cues. Extinguished and reinstated rats self-administered similar amounts of sucrose over the course of self-administration (A, inset, Student’s t(8) = 0.6214, p = 0.5516). B Reinstated rats pressed more during 15 min of cue exposure than 15 min in the extinguished context during the last extinction session (Kolmogorov–Smirnov = 1.00, **p = 0.0079). C GAT-3 expression by dlVP astroglia was unchanged during extinction training but increased during cued reinstatement (Kruskal–Wallis = 7.699, p = 0.0213, *p = 0.0168 vs. Yoked using Dunn’s post hoc test). D SYNJ1 co-registration by dlVP astroglia was also elevated during cued reinstatement of sucrose seeking (Kruskal–Wallis = 9.323, p = 0.0095, #p = 0.0176 vs. Ext using Dunn’s post hoc test). Data shown as mean ± SEM (A, B) or median (C, D). N is shown in legend as cells/animals (C, D). Yoked yoked cues, Ext extinguished, Rst 15-min reinstated.
Fig 5: Tonic GABA currents in striatal spiny projection neurons (SPNs) are augmented by GAT inhibition.a, b, d, e Left, representative continuous whole-cell recordings from SPNs in DLS (a, d, e) or NAcC (b) voltage clamped at −70 mV in the presence of ionotropic glutamate receptor antagonists NBQX (5 μM) and D-AP5 (50 μM), before and during bath application of GAT inhibitor NPA (blue, 1.5 mM, n = 7 cells/5 mice for DLS in a, n = 6 cells/3 mice for NAcC in b), GAT-1-specific inhibitor SKF89976A (orange, 20 μM, n = 6 cells/3 mice in d), or the combined application of SKF89976A and GAT-3-specific inhibitor SNAP5114 (green, 50 μM, n = 6 cells/4 mice in e). GAT inhibitors increase the extracellular GABAA-mediated inward current, revealed by a shift in the holding current, and is reversed upon application of GABAA receptor antagonist picrotoxin (PTX, 100 μM). Right, mean holding current in pA recorded in SPNs in control conditions, upon addition of GAT inhibitors and then PTX. c, f Mean tonic GABAA-receptor-mediated currents induced by GAT inhibition recorded from SPNs, calculated by subtracting predrug holding current from GAT block-induced holding current. Friedman’s ANOVA on Ranks and Student–Newman–Keuls multiple comparisons (a, b, d, e), Mann–Whitney U test (c), Kruskal–Wallis test and Dunn’s multiple comparisons (f). *p < 0.05, **p < 0.01. Error bars are ±SEM. Source data are provided as a Source Data file.
Supplier Page from Abcam for Anti-GABA Transporter 3 / GAT 3 antibody