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. 2017 Nov 9;13(11):e1007059.
doi: 10.1371/journal.pgen.1007059. eCollection 2017 Nov.

A subset of sweet-sensing neurons identified by IR56d are necessary and sufficient for fatty acid taste

Affiliations

A subset of sweet-sensing neurons identified by IR56d are necessary and sufficient for fatty acid taste

John M Tauber et al. PLoS Genet. .

Abstract

Fat represents a calorically potent food source that yields approximately twice the amount of energy as carbohydrates or proteins per unit of mass. The highly palatable taste of free fatty acids (FAs), one of the building blocks of fat, promotes food consumption, activates reward circuitry, and is thought to contribute to hedonic feeding underlying many metabolism-related disorders. Despite a role in the etiology of metabolic diseases, little is known about how dietary fats are detected by the gustatory system to promote feeding. Previously, we showed that a broad population of sugar-sensing taste neurons expressing Gustatory Receptor 64f (Gr64f) is required for reflexive feeding responses to both FAs and sugars. Here, we report a genetic silencing screen to identify specific populations of taste neurons that mediate fatty acid (FA) taste. We find neurons identified by expression of Ionotropic Receptor 56d (IR56d) are necessary and sufficient for reflexive feeding response to FAs. Functional imaging reveals that IR56d-expressing neurons are responsive to short- and medium-chain FAs. Silencing IR56d neurons selectively abolishes FA taste, and their activation is sufficient to drive feeding responses. Analysis of co-expression with Gr64f identifies two subpopulations of IR56d-expressing neurons. While physiological imaging reveals that both populations are responsive to FAs, IR56d/Gr64f neurons are activated by medium-chain FAs and are sufficient for reflexive feeding response to FAs. Moreover, flies can discriminate between sugar and FAs in an aversive taste memory assay, indicating that FA taste is a unique modality in Drosophila. Taken together, these findings localize FA taste within the Drosophila gustatory center and provide an opportunity to investigate discrimination between different categories of appetitive tastants.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Gr64f gustatory receptor neurons respond to sucrose and HxA.
(A) Expression of GFP in Gr64f-GAL4 neurons reveals axon terminals in the sub-esophagael zone (SEZ). Scale bar = 50 μm. (B) Diagram of live-imaging experimental protocol. Cuticle above the SEZ is removed and GCaMP5 fluorescence is recorded while tastant is applied to the proboscis. (C) Average peak %ΔF/F during response to water, 1% HxA, and 10mM sucrose (n = 11, 11, 10 respectively). Error bars indicate SEM. One-way ANOVA with Tukey’s HSD; ***p<0.001. (D) Average %ΔF/F traces and representative images of calcium activity in Gr64f neurons responding to water, (E) 1% HxA, and (F) 10mM sucrose. Scale bar = 50 μm. Shaded region of trace indicates +/- SEM.
Fig 2
Fig 2. IR56d neurons are necessary and sufficient for PER to FAs.
(A) Expression pattern of IR56d neurons visualized with GFP. Distinct regions of projection are seen in (B) posterior and (C) anterior regions of SEZ. Scale bar = 50 μm. (D) Blocking synaptic release by genetic expression of light-chain tetanus toxin (TNT) in IR56d neurons significantly reduces PER to HxA, but not sucrose, compared to control flies expressing an inactive form of tetanus toxin (impTNT). (impTNT n = 26; TNT n = 29). Wilcoxon Rank Sum Test; ***p<0.001. (E) Heat activation of IR56d neurons with TRPA1 induces significant PER compared to either transgene alone, and is comparable to PER induced by activation of Gr64f neurons. (n = 49 for all genotypes). Fisher’s Exact Test with Bonferroni correction for multiple comparisons. (F) Targeted knockdown of norpA in IR56d neurons significantly reduces response to HxA, while response to sucrose did not differ from controls flies harboring IR56d-GAL4 alone (N>27 for all genotypes); ***p< 0.001.
Fig 3
Fig 3. Response to sugar and fatty acid differs in anterior and posterior IR56d projections.
(A) Activity traces and representative images of calcium activity in IR56d posterior projections in response to water, 1% HxA, and 10mM sucrose (n = 13 for each tastant). Shaded region of trace indicates +/- SEM (B) Average peak %ΔF/F for data shown in (A). Error bars indicate SEM. One-way ANOVA with Tukey’s HSD; **p< 0.01, ***p< 0.001. (C) Activity traces and representative images of calcium activity in IR56d posterior projections in response to water, 1% HxA, and 10mM sucrose (n = 12 for each tastant). Shaded region of trace indicates +/- SEM (D) Average peak ΔF/F for data shown in (C). Error bars indicate SEM. One-way ANOVA with Tukey’s HSD; ***p< 0.001.
Fig 4
Fig 4. IR56d neurons are selectively responsive to short and medium-chain FAs.
(A) PER in response to short-chain pentanoic acid (PeA, n = 46), medium-chain octanoic acid (OcA, n = 26), and long-chain oleic acid (n = 46) in TNT and control impTNT flies. Blocking synaptic release in IR56d neurons with TNT significantly decreases PER to octanoic acid (n = 29), but does not affect PER for pentanoic (n = 42) or oleic acid (n = 42). Wilcoxon Rank-Sum Test; ***p<0.001. (B) Average peak %ΔF/F for the posterior projections of IR56d neurons in response to water, 1% DMSO, pentanoic acid, octanoic acid, and oleic acid (n = 9, 8, 9, 9, 8, respectively), and (C) the anterior projections (n = 9, 7, 9, 9, 7, respectively). Error bars indicate SEM. One-way ANOVA with Tukey’s HSD; ***p<0.001.
Fig 5
Fig 5. IR56d anterior projections are dispensable for PER to FAs.
(A) IR56d and Gr64f neurons visualized with IR56d-GAL4 driving RFP and Gr64f-LexA driving GFP. Co-localization is detected in the posterior projections, but is minimal in anterior projections. (B) Driving the GAL4 repressor Gal80 with Gr64f-LexA limits GFP expression to the non-overlapping IR56d anterior projections. (C) Restricting TNT expression to the non-overlapping anterior projection neurons does not significantly impact PER to sugar or HxA (impTNT n = 23; TNT n = 22). Error bars indicate SEM. Wilcoxon Rank Sum Test Sucrose: p>0.98; HxA: p>0.96. (D) Restricting TRPA1 expression to the non-overlapping neurons abolishes PER compared to control in which TRPA1 is driven in all IR56d neurons. Fisher’s Exact Test with Bonferroni correction for multiple comparisons; ***p<0.001.
Fig 6
Fig 6. Drosophila discriminates between sucrose and HxA.
(A) Taste memory protocol to determine sucrose and FA discrimination. Flies are trained by pairing HxA or sucrose on tarsi with quinine on proboscis. PER in response to HxA and sucrose is then tested following training to sucrose or HxA in the absence of quinine. In control experiments (naïve), the same procedure is followed, but quinine is not applied to the proboscis. (B) The pairing of sucrose and quinine (dark green circles) results in a significant reduction in PER across all three training trials compared to unpaired naïve flies (light green circles). PER response to sucrose in the test is significantly lower in trained flies compared to naïve flies (n = 7, 11), but no differences in response to HxA (triangles) is detected between the experimental and naïve groups (n = 11, 12). Kruskal-Wallis Test followed by Dunn’s Test (control: w1118); **p<0.01. (C) The pairing of HxA and quinine (dark blue triangles) results in a significant reduction in PER across all three training trials compared to unpaired naïve flies (light green triangles). The test PER response to HxA alone is significantly lower in trained flies compared to naïve flies (n = 13, 16), but no differences in response to sucrose (circles) is detected between the groups (n = 12, 12). Kruskal-Wallis Test followed by Dunn’s Test (control: w1118); **p<0.01. (D) Percent suppression of PER reveals that flies trained and tested to the same tastant (either sucrose or HxA) show significantly reduced PER compared to flies trained and tested with different tastants.

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