Figure 4.

TyrR^PLP neurons respond to tyramine and sucrose in starved males

(A and B) Application of 1 mM tyramine (middle panel) leads to a decrease in the Ca²⁺ response of TyrR^PLP neurons only in starved (blue, A) but not fed (red, B) males, in comparison to the response before the application (left panel). After 5 min of washout with saline (right panel) responses return to baseline. The ΔF/F₀ represents the evoked fluorescence change from baseline. Traces were averaged from 11–16 flies. Solid line represents the mean and shaded areas indicate SEM. Genotype tested: TyrR-GAL4/UAS-GCaMP6s;61A01-GAL80.

(C and D) Quantification of mean ΔF/F₀ responses of TyrR^PLP neurons over 90 s before (pre) and after the application of 1 mM tyramine in starved (blue, C) and fed (red, D) males. In starved males, a significant reduction of the mean Ca²⁺ response to 1 mM tyramine was observed (n = 16). In fed males, no changes were apparent (n = 11).

(E and F) Application of 1 mM of tyramine (middle panel) hyperpolarizes TyrR^PLP neurons in starved (E, blue) males, as indicated by increase in fluorescence (decrease in −ΔF/F₀) of the ASAP2 voltage sensor in comparison to the response before application (left panel). In fed (F, red) males no changes were observed. The −ΔF/F₀ represents the evoked fluorescence change from baseline. Traces were averaged from 8–9 flies. The voltage-imaging plot fluorescence changes are plotted on a −ΔF/F₀ scale to correct for the inverse relation between membrane voltage and sensor fluorescence. Genotype tested: UAS-ASAP2/61A01-LexA;LexAopGa80/TyR-GAL4.

(G and H) Quantification of the mean −ΔF/F₀ responses of TyrR^PLP neurons over 90 s before (pre) and after the application of 1 mM tyramine in starved (G, blue) and fed (H, red) males (using ASAP2). Changes to 1 mM tyramine were observed in starved (n = 9) but not fed males (n = 8).

(I) Application of 1 mM tyramine (right panel) in starved males expressing TAR3 RNAi in TyrR neurons did not change the mean Ca²⁺ response in comparison to baseline (left panel). Traces were averaged from 9 animals. Genotype tested: UAS-GCaMP6s/+;TyR-GAL4/UAS-TAR3 RNAi.

(J) Quantification of mean ΔF/F₀ responses of TyrR^PLP neurons in starved males expressing TAR3 RNAi over 90 s before (pre) and after application of Tyr (1 mM tyramine). No changes were observed in the mean Ca²⁺ response to 1 mM tyramine (n = 9).

(K) In starved males, feeding on 100 mM sucrose leads to a Ca²⁺ increase in TyrR^PLP neurons in comparison to the response before sugar consumption (pre). Traces were averaged from 11 flies. Only flies that were confirmed to ingest sucrose were included in the analysis (n = 11). Genotype tested: TyrR-GAL4/UAS-GCaMP6s;61A01-GAL80/+.

(L) Quantification of mean ΔF/F₀ responses of TyrR^PLP before (pre; 16 s) and during sugar consumption (60 s).

(M) UAS-mCD8-GFP expressed with TyrR-GAL4 in the adult brain. Anti-GFP (green) and anti-AstC (magenta) staining is shown. Magnified images of the lateral protocerebrum show clear overlap between TyrR^PLP cells and anti-AstC. SMP, superior medial protocerebrum; PLP, posterior lateral protocerebrum; IPS, inferior posterior slope; GNG, gnathal ganglia.

(N) UAS-mCD8-GFP expressed with AstC-GAL4 (II) in the adult brain. Anti-GFP and anti-AstC. Scale bars, 50 or 25 μm, insets.

(O) Courtship preference indices of 24-h starved AstC-GAL4 (II)/UAS-TNT males and the genetic controls (n = 26–29).

(P) Working model. TyrR^PLP neurons are antagonistically regulated by tyramine and food detection.

Lines show the mean and error bars indicate SEM. All boxplots represent 25^th to 75^th percentile and black line as the mean. ^∗p < 0.05 calculated using paired t test in (C) and (D); Wilcoxon matched-pairs signed rank test in (G), (H), (J), and (L); and Kruskal Wallis test followed by Dunn’s post hoc multiple comparison in (O). Absence of asterisk (^∗) denotes non-significant data.

See also Figure S3.