Supplementary Figure 3: Effects of artificially increasing dopamine levels or ablating dopamine-excitable neurons in striatum

A. Schematic representation of the behavioural preparation where mice licked bitter-containing sippers such that detected licks triggered intra-gastric infusions of either glucose or sucralose. B. Hungry mice (N=8) licked the bitter solution significantly more when self-infusing glucose compared to when self-infusing sucralose (t[7]=2.62, * p=0.035), resulting in significantly larger intra-gastric glucose volumes (not shown, t[7]=3.17, p=0.016). C. Reverse microdialysis was used to perfuse DS or VS with dopamine during ingestion of sucralose or a bitter solution. D. Dopamine perfusion in DS (N=6) and VS (N=5) resulted in increases in sucralose intake when compared to aCSF perfusions (Left panel; Perfusion main effect F[1,9]=16.76, p=0.003) The effect was similar in both VS and DS (Two-way RM-ANOVA, striatal region × brain perfusion F[1,9]=3.43, p=0.097; dopamine vs. aCSF in DS, t[5]=5.07, Bonferroni * p=0.008; in VS effect was weaker: t[4]=1.33, p=0.51). This is consistent with sweetness-driven dopamine efflux in VS but not DS. Dopamine perfusion in DS and VS resulted in robust increases in bitter intake when compared to aCSF perfusions (Right panel; Perfusion main effect F[1,9]=27.55, p=0.001). Effect was similarly robust in both VS and DS (Two-way RM-ANOVA, striatal region × brain perfusion F[1,9]=0.43, p=0.53; dopamine vs. aCSF in DS, t[5]=3.6, Bonferroni *p=0.03; VS: t[4]=3.7, ** p=0.04). E. Cell-specific ablation of D1r-neurones in DS or VS. Top panel: Brief access test for different sucralose concentrations. [Two-way RM-ANOVA, sweetness × group F[6,57]=8.23, p=0.000002; group effect F[2,19]=1.2, p=0.32;; sweetness effect F[3,57]=95.8, p= 3x10^-22]. Cell-specific ablation of D1r-neurones in VS, but not DS, produced a lower intake of 2 mM sucralose [One-way ANOVA group effect F[2,21]=5.95, p=0.01. Ventral lesion vs. Control, Bonferroni *p=0.04; and vs. DS lesion, Bonferroni * p=0.014], whereas at 6mM sucralose VS Casp intake was higher [One-way ANOVA group effect F[2,21]=5.94, p=0.01; ventral lesion vs. WT-Casp Bonferroni #p=0.012 and Ventral lesion vs. DS-Casp Bonferroni p=0.055]. Bottom panel: Masking bitterness. The concentration of the bitter compound denatonium was fixed at 6mM and different concentrations of sucralose were used. [Two-way RM-ANOVA, sweetener concentration × group F[6,57]=5.29, p=0.00021; group effect F[2,19]=1.95, p=0.17; sweetener concentration effect F[3,57]=67.32,, p=9.9x10^-19]. Cell-specific ablation of D1r-neurones in VS, but not DS, produced a lower intake of the mixture 6mM denatonium and 6mM sucralose [One-way ANOVA group effect F[2,21]=7.55, p=0.004. Ventral lesion vs. Control, Bonferroni **p=0.012; and vs.DS lesion, Bonferroni ** p=0.007]. F. Effects of cell-specific ablation of D1r-neurones in DS or VS on sugar-driven consumption of unpalatable solutions. Top panel: Licks produced during the ingestion of a bitter mixture that triggers intra-gastric infusions of different D-glucose concentrations. [Two-way RM-ANOVA, glucose concentration × group F[6,57]=2.68, p=0.023; group effect F[2,19]=2.75, p=0.089; glucose concentration effect F[3,57]=25.8, p=1.3x10^-10]. Bottom panel: Intra-gastric infusions observed during these sessions [Two-way RM-ANOVA, glucose concentration × group F[6,57]=8.83, p=8.2x10^-7; group effect F[2,19]=4.18, p=0.031; glucose concentration effect F[3,57]=45.87, p=3.3x10^-15]. Concentrations: 0.5% [One-way ANOVA group effect F[2,21]=7.07, p=0.005; ventral lesion vs. Control, Bonferroni *p=0.007; and vs.DS lesion, Bonferroni * p=0.023];: 10% [One-way ANOVA group effect F[2,21]=7.09, p=0.005; intake in dorsal lesion vs. Control, Bonferroni **p=0.007; and vs.VS lesion, Bonferroni ** p=0.024]; 25% [One-way ANOVA group effect F[2,21]=5.75, p=0.011; dorsal lesion vs. Control, Bonferroni #p=0.034; and vs.VS lesion, Bonferroni #p=0.018]; 50% [One-way ANOVA group effect F[2,21]=2.16, p=0.142]. G. Licks (top) and infusions (bottom) produced during the conditioning sessions (bitter intake paired with intra-gastric infusions of glucose) prior to the second two-bottle test. Cell-specific ablation of D1r-neurones did not alter intake. [Licks: One-way ANOVA Group Effect F[2,21]=2.27, p=0.13; Infusions: One-way ANOVA Group Effect F[2,21]=1.11, p=0.34]. H-K. Neuroanatomical analyses of the sham cell-specific lesions. When Retrobeads were injected into globus pallidus (GP, targeted by D2r-expressing neurons of DS) of DS-CTL mice (area within dotted line in H), strong labelling was observed in DS (I). Similarly, robust labelling was observed in DS (J) when Retrobeads were injected into the contralateral SNr (K), which is exclusively targeted by D1r-expressing neurons of DS (confront vs. Main Figure 2 in which lesioned case is shown). To allow visualization of the relevant anatomical landmarks, images show the Retrobead fluorescence signal overlaid on a bright field image of the same section.

Abbreviations: DS-Casp: Caspase-driven D1r-dependent lesions in DS of D1r-Cre mice (N=7); VS-Casp: Caspase-driven D1r-depedent lesions in VS of D1r-Cre mice (N=7); WT-Casp: Viral delivery of Cre-dependent caspase in DS and/or VS of non-Cre mice (N=8). n.s. = non-statistically significant.

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