Branch-specific plasticity of a bifunctional dopamine circuit encodes protein hunger

Abstract

Free-living animals must not only regulate the amount of food they consume but also choose which types of food to ingest. The shifting of food preference driven by nutrient-specific hunger can be essential for survival, yet little is known about the underlying mechanisms. We identified a dopamine circuit that encodes protein-specific hunger in Drosophila The activity of these neurons increased after substantial protein deprivation. Activation of this circuit simultaneously promoted protein intake and restricted sugar consumption, via signaling to distinct downstream neurons. Protein starvation triggered branch-specific plastic changes in these dopaminergic neurons, thus enabling sustained protein consumption. These studies reveal a crucial circuit mechanism by which animals adjust their dietary strategy to maintain protein homeostasis.

Fig. 1. DA-WED neurons are necessary and sufficient for protein hunger

(A) Preference Index (PI) for yeast vs sucrose in mated female flies with indicated genotypes (n=5–15 trials). (B) Whole-mount brain immunostaining with anti-GFP (green) and anti-Bruchpilot (BRP, nc82, magenta). Scale bar: 100µm. High-magnification sections of DA-WED neuron cell bodies are shown on the right with anti-GFP (green) and anti-TH (blue). Scale bar: 10 µm. (C) Whole-mount brain immunostaining with anti-GFP (green) and anti-DsRed (magenta). “M” and “L” denote Medial (M) and Lateral (L) branches, respectively. Scale bar: 50 µm. (D and E) PI (D) and yeast intake per fly (E) showing that silencing DA-WED neurons suppressed protein hunger in yeast-deprived male flies (n=8–18 and 18–33 trials for D and E). (F and G) Conditional activation of DA-WED neurons increased preference for (F) and intake of (G) yeast in male flies (n=7–13 and 12–27 trials for F and G). Simplified box plots show 25^th, 50^th, and 75^th percentiles. For bar graphs, mean ± SEM is shown. In this and subsequent figures “*”, “**”, “***”, and “ns” denote P<0.05, P<0.01, P<0.001, and not significant, respectively.

Fig. 2. Protein starvation increases activity of DA-WED neurons

(A and B) Yeast deprivation increased spontaneous action potential (AP) firing rate from DA-WED neurons (n=7–9). (C to E), Mean AP frequency and f-I slope for evoked responses from DA-WED neurons was elevated following protein starvation (n=5). All recordings were from male flies. (F) Representative images of DA-WED neuron cell bodies from control, yeast-deprived, and yeast-deprived with amino acid supplemented TH-D-Gal4>CaLexA male flies. Scale bar: 5 µm. (G) CaLexA signal intensity from DA-WED cell bodies correlated with protein hunger status (n=12–18). (H and I) An amino acid mix lacking Gln failed to suppress the increase in DA-WED activity (n=4–5) and was less effective at inhibiting protein consumption following yeast deprivation (n=17–25 trials) in male flies.

Fig. 3. Yeast and sucrose feeding are oppositely regulated by DA-WED neurons

(A) Silencing DA-WED neurons increased sucrose intake in protein-deprived male flies (n=12–34 trials). (B) Conditional activation of DA-WED neurons reduced sucrose consumption in male flies (n=21–33 trials). (C) Knockdown of DopR2 using the drivers shown reduced yeast intake in protein-deprived male flies (n=18–47 trials). (D) Knockdown of DopR1 using the drivers shown elevated sucrose intake in protein-deprived male flies (n=15–40 trials). (E and G) Representative image from a GRASP experiment between DA-WED neurons and neurons labeled by R70G12-Gal4 or R72B03-Gal4. Native fluorescence from reconstituted GFP is shown. (F and H) Whole-mount brain image from Multicolor Flip-Out (MCFO) analysis for the driver lines shown stained with anti-HA (green) and anti-Bruchpilot antibodies (magenta). Brains in E–H are from male flies. Scale bar: 100µm.

Fig. 4. Protein starvation triggers branch-specific plastic changes in DA-WED cells

(A to C) DA-WED neuron medial, but not lateral, branch length and Syt+ puncta number were greater in yeast-deprived vs control male flies, and these effects were suppressed by Gln (n=17–20 for B and 25–56 for C). Scale bar: 50µm. (D to F) Sharp intracellular recordings of postsynaptic potentials (PSPs) from FB-LAL neurons in male flies (n=6–11). Yeast deprivation increased PSP frequency and induced a population of high amplitude PSPs, which was suppressed by knockdown of DopR2 in these neurons. (G to I) The increased yeast intake (G) and Syt+ puncta number in the medial branch (I), but not decreased sucrose intake (H), induced by activation of DA-WED neurons persisted for at least 6 hrs following cessation of heat treatment in male flies (n=7–25 trials for G and 9–16 trials for H). Scale bar: 25µm. (J) Model. At baseline (top), circulating Gln levels are high and act directly or indirectly on DA-WED neurons to suppress their activity, leading to greater sugar intake. Severe protein deprivation (bottom) reduces Gln levels, thus activating the DA-WED cells. Under these conditions, the medial branch (magenta) undergoes plastic changes, resulting in increased and persistent stimulation of the downstream FB-LAL neurons via DopR2 receptors to promote prolonged protein intake. Simultaneously, signaling from the lateral branch to downstream PLP neurons via DopR1 receptors induces a transient inhibition of sugar intake. Together, these changes result in a behavioral switch from consuming sugar to persistent intake of protein.