Extended Data Fig. 12: Increased striatal dopamine suppresses food intake.

(a) Schematic depicting hM3D(Gq) expression in the VTA of a DAT::Cre mouse, and GRAB^DA expression and fibre implant in the ventral striatum. (b-c) Representative images of hM3D(Gq) expression in the VTA (b), and GRAB^DA expression and fibre track in the ventral striatum (c) of a DAT::Cre mouse. Scale bar, 500 µm in B, 200 µm in C. (d) Average ∆F/F of GRAB^DA signals in 3-min bins following VTA neuron activation with vehicle and varying concentrations of CNO (0.025 mg/Kg, 0.25 mg/Kg, 1 mg/Kg and 2.5 mg/Kg; n = 8 per group, repeated measures two-way ANOVA interaction P < 0.001, main effect P < 0.001; Holm-Sidak’s). (e) Net area under curve ∆F/F of GRAB^DA signals following VTA neuron activation with vehicle and varying concentrations of CNO (0.025 mg/Kg, 0.25 mg/Kg, 1 mg/Kg and 2.5 mg/Kg; n = 8 per group, repeated measures one-way ANOVA P < 0.001; Holm-Sidak’s, P = 0.012 (vehicle versus 0.025 mg/Kg), P=0.010 (vehicle versus 0.25 mg/Kg), P = 0.023 (vehicle versus 1.0 mg/Kg), P = 0.010 (vehicle versus 2.5 mg/Kg)). (f) Food intake of food-deprived mice following VTA neuron activation with vehicle and varying concentrations of CNO (0.025 mg/Kg, 0.25 mg/Kg, 1 mg/Kg and 2.5 mg/Kg; n = 8 per group, repeated measures one-way ANOVA P<0.001; Holm-Sidak’s, P < 0.001 (vehicle versus 0.025 mg/Kg), P < 0.001 (vehicle versus 0.25 mg/Kg), P < 0.001 (vehicle versus 1.0 mg/Kg), P < 0.001 (vehicle versus 2.5 mg/Kg)). (g) Plot of GRAB^DA signals and corresponding food intake in food-deprived mice treated following VTA neuron activation (n = 8 per group, Pearson correlation). Solid line indicates the linear trend line fit to the data. (h) Average ∆F/F of GRAB^DA signals from 0 to 30 min following treatment with vehicle and varying concentrations of CNO (n = 8 per group, repeated measures one-way ANOVA P < 0.001; Holm-Sidak’s, P = 0.004 (vehicle versus 0.025 mg/Kg), P = 0.004 (vehicle versus 0.25 mg/Kg), P = 0.004 (vehicle versus 1.0 mg/Kg), P = 0.004 (vehicle versus 2.5 mg/Kg)). (i) Maximum ∆F/F of GRAB^DA signals following treatment with vehicle and varying concentrations of CNO (n = 8 per group, repeated measures one-way ANOVA P=0.023; Holm-Sidak’s, P = 0.034 (vehicle versus 2.5 mg/Kg)). (j) Average ∆F/F of GRAB^DA signals during presentation of food in fasted mice following treatment with vehicle and varying concentrations of CNO (0.025, 0.25, 1.0 and 2.5 mg/Kg). Signals are aligned to food presentation. Dark lines represent mean values and lighter shaded areas represent SEM (n = 8). (k) Heatmaps reporting ∆F/F of GRAB^DA signals in individual mice in (j) (n = 8). (l) Maximum ∆F/F of GRAB^DA signals during food presentation in mice following treatment with vehicle and varying concentrations of CNO (n = 8 per group, one-way ANOVA P = 0.0117; Holm-Sidak’s, P = 0.0389 (vehicle versus 0.25 mg/Kg), P = 0.0056 (vehicle versus 1.0 mg/Kg), P=0.0121 (vehicle versus 2.5 mg/Kg)). (m) Scatter plot depicting the maximal ∆F/F GRAB^DA response to food following pre-stimulation of VTA DA neurons and the associated amount of food intake following pre-stimulation of VTA DA neurons (n = 8 per group, Pearson correlation, P < 0.01). Solid line shows the linear trend line fit to the data. (n-p) Images of hM4D(Gi) expression (red) in TH⁺, VTA neurons (green) of a DAT::Cre mouse (n). Higher magnification of white box (o-p). Scale bar, 500 µm (n), 50 µm (p). (q) Neurons transduced with hM4D(Gi) in the VTA and SNC (n = 3, 1047, 2745, 2710 neurons each mouse, unpaired t-test, P = 0.02). (r) Average ∆F/F of DA signals in aDCN-LAT hM3D(Gq) mice and aDCN-LAT hM3D(Gq); VTA hM4D(Gi) mice (n = 6 per group, unpaired t-test, P = 0.003). (g) Distance travelled by aDCN-LAT hM3D(Gq) mice and aDCN-LAT hM3D(Gq); VTA hM4D(Gi) mice during a 10-min open field session (n = 6 and 7, respectively, unpaired t-test, P = 0.382). Data are expressed as mean ± SEM, two-sided P values, t-tests and post-hoc comparisons: *P < 0.05, **P<0.01, ***P < 0.001; ANOVA interaction: ∞∞∞P < 0.001; ANOVA main effect of group: ¤¤¤P < 0.001. SNC, substantia nigra pars compacta; TH, tyrosine hydroxylase; VTA, ventral tegmental area. Statistical analysis in Supplementary Table 1.

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