A Neural Circuit Arbitrates between Persistence and Withdrawal in Hungry Drosophila

Abstract

In pursuit of food, hungry animals mobilize significant energy resources and overcome exhaustion and fear. How need and motivation control the decision to continue or change behavior is not understood. Using a single fly treadmill, we show that hungry flies persistently track a food odor and increase their effort over repeated trials in the absence of reward suggesting that need dominates negative experience. We further show that odor tracking is regulated by two mushroom body output neurons (MBONs) connecting the MB to the lateral horn. These MBONs, together with dopaminergic neurons and Dop1R2 signaling, control behavioral persistence. Conversely, an octopaminergic neuron, VPM4, which directly innervates one of the MBONs, acts as a brake on odor tracking by connecting feeding and olfaction. Together, our data suggest a function for the MB in internal state-dependent expression of behavior that can be suppressed by external inputs conveying a competing behavioral drive.

Keywords: DopR2; Drosophila melanogaster; dopamine; foraging; goal-directed behavior; learning; mushroom body; octopamine; olfactory system; persistence.

Graphical abstract

Figure 1

Persistent Odor Tracking Is Motivated by Hunger (A) Top: Spherical-treadmill assay for olfactory stimuli. (B) Top: Average running speed with SEM (mm/s) of 18 wild-type Canton S flies under repeated vinegar exposure for 10 trials. Shaded areas represent the odor exposure duration. Bottom: Average running speeds of flies during vinegar exposure were significantly higher compared to the speeds observed during pre- and post-stimulation periods. (C) Top: Average absolute turning speed with SEM (deg/s) of 18 flies under repeated vinegar exposure for 10 trials. Bottom: The absolute turning speed under vinegar was significantly lower than the turning speed recorded in pre- and post-stimulation periods. (D) Average running speed with SEM of 18 wild-type flies over time for each of the individual 10 trials. (E) Comparison of average running over trials. (F) Average running speed with SEM of fed (0 h) and hungry (24 and 48 h) flies during trial 1 and trial 10 of repeated vinegar exposure. (G and H) Average running speed with SEM during odor stimulation during trial 1 to 10 for fed, 24 h and 48 h starved flies. The boxplot (G) displays the Tukey’s post hoc analysis for the main group effect. (I and J) Average running bout times with SEM during open-loop odor exposure for fed and food-deprived flies over 10 trials. The boxplot (J) displays the Tukey’s post hoc analysis for the main group effect. (K) Left: Schematics for the closed-loop assay. Right: Average running bout times with SEM during closed-loop odor exposure for differentially food-deprived flies over 10 trials. (L) Total running times of fed and starved flies during odor stimulation. (M) Average summed running bout times during 10 trials for all groups in closed-loop experiments under vinegar exposure. For all analyses, statistical notations are as follows: ns, p > 0.05; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. In all panels, error bars denote SEM.

Figure 2

Two Synaptically Connected MBONs Are Required for Odor Tracking (A) Running speeds during stimulus phase in trial 1 and trial 10 upon blocking synaptic output of MVP2/MBON-γ1pedc>αβ (MB112C>UAS-Shibire^ts1) at non-permissive temperature compared to control with empty-Gal4 (pBDPU-Gal4>UAS-Shibire^ts1). (B) Average running speeds during stimulus phase over trials. (C) Running speeds during acute MBON-γ1pedc>αβ activation with CsChrimson (MB112-Gal4>UAS-CsChrimson) in starved flies compared to controls (pBDPU-Gal4>UAS-CsChrimson). (D) Running speeds during stimulus phase over trials. (E) Running speeds during stimulus phase in trial 1 and trial 10 upon blocking synaptic output of MBON-α2sc (MB80C) compared to controls (pBDPU-Gal4>UAS-Shibire^ts1). (F) Running speeds during stimulus phase over trials. (G) Running speeds in starved flies when the MB80C neuron was activated via optogenetics (MB80C>UAS-CsChrimson) (controls: pBDPU-Gal4>UAS-CsChrimson). (H) Running speeds during stimulus phase over trials. For all analyses, statistical notations are as follows: ns, p > 0.05; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. In all panels, error bars denote SEM.

Figure 3

MBONs Show Trial- and Feeding State-Dependent Odor Responses (A) Left: Scheme of MBON-γ1pedc>αβ (MB112C) innervating the MB lobes. Right: MB112C>GCaMP6f expression in an in vivo two-photon preparation. ROIs are marked by dashed lines (scale bar 10 μm). (B) Average traces of odor responses (purple line) of MB112C peduncle dendrites from trial 1 to 10. (C) Average traces of odor responses of MB112C γ1 dendrites from trial 1 to 10. (D) Quantification of the area under the fluorescence response during the stimulus period in the peduncle region from trial 1 to 10. (E) Quantification of the area under the fluorescence response during the stimulus period in the γ1 region from trial 1 to 10. (F) Left: Scheme of MBON-α2sc (MB80C) innervating the MB lobes. Right: MB80C>GCaMP6f expression in an in vivo two-photon preparation. The ROI is marked by dashed lines (scale bar 10 μm). (G) Average traces of odor responses of MB80C axons from trial 1 to 10. (H, I) Quantification of the area under the fluorescence responses during the stimulus period and after the stimulus period. (J) Quantification of the offset peak decay time constant over trials. For all analyses, statistical notations are as follows: ns, p > 0.05; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. In all panels, error bars denote SEM.

Figure 4

Taste and Food Suppress Odor Tracking (A) Average running speeds of 24 h and 24 h, 30 min re-fed animals at trial 1 and trial 10. (B) Running speeds over trials in 24 h starved and 30 min re-fed flies compared to 24 h starved animals. The boxplot displays the main group effect. (C) Left: Schematics of the concurrent odor and optogenetic-activation protocol. Right: Gr5a>UAS-CsChrimson and Gr43a>UAS-CsChrimson flies compared to control (pBDPU-Gal4>UAS-CsChrimson). (D) Running speed over trials during simultaneous odor and light activation. The boxplot displays the main group effect. (E) Average running activity displayed as boxplots for flies expressing CsChrimson in Gr5a or Gr43a neurons (Gr5a-Gal4;UAS-CsChrimson and Gr43a-Gal4;UAS-CsChrimson). For all analyses, statistical notations are as follows: ns, p > 0.05; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. In all panels, error bars denote SEM.

Figure 5

A Subset of Octopaminergic Neurons Inhibit Odor Tracking (A) Acute optogenetic activation of octopaminergic neurons. CsChrimson was expressed in octopaminergic neurons by Tdc2>UAS-CsChrimson (Controls: UAS Ctrl: +>UAS-CsChrimson, Gal4 Ctrl: Tdc2-Gal4>+). Running speeds during trial 1 and trial 10. (B) Evolution of average running speeds for Tdc2>UAS-CsChrimson flies during odor exposure over trials. The boxplot displays the main group effect. (C) Acute optogenetic activation of VPM neurons. MB22B harbors VPM3 and VPM4 neurons, whereas MB113C labels only VPM4 (Control: pBDPU-Gal4>UAS-CsChrimson). Running speeds during trial 1 and trial 10. (D) Average running speeds for MB22B>UAS-CsChrimson and MB113C>UAS-CsChrimson flies during odor exposure. The boxplot displays the main group effect. (E) Left: Scheme of optogenetic and olfactory behavioral test arena. Right: Average preference index during optogenetic activation of octopaminergic neurons under vinegar exposure. (F) Activation of VPMs compared to genetic controls. (G–J) Expression patterns and polarity of MB22B (H, H′, and J) and MB113C (G, G′, and J) split-Gal4 lines. SEZ (subesophageal zone); PEZ (periesophageal zone) (K–L″) VPM4 neurons (MB113C>mCD8GFP) express octopamine. For all analyses, statistical notations are as follows: ns, p > 0.05; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. In all panels, error bars denote SEM.

Figure 6

VPM4 Modulates MVP2-Dependent Tracking (A–C) EM reconstruction reveals synaptic connections between MBON-γ1pedc>αβ and VPM3 and VPM4. (A) Skeletons of EM reconstruction of MBON-γ1pedc>αβ (blue), VPM3 (red), and VPM4 (purple) on the neuropil of a whole fly brain. (B) Red (VPM3) and purple (VPM4) indicate the synapses between VPMs and MBON-γ1pedc>αβ, respectively. (C) Higher magnification of B. (D) Scheme showing VPM4 and MBON-γ1pedc>αβ neurons at the level of the mushroom body and the genetic combination of transgenes expressed in the fly used for the experiment. (E) Left: Average traces of GCaMP fluorescence in MBON-γ1pedc>αβ dendrites upon ATP application on brain in an in vivo preparation. Right: Boxplots displaying peak amplitude of % ρF/F GCaMP fluorescence in MBON-γ1pedc>αβ/MVP2 upon ATP application. (F) Left: Representative pseudocolored images displaying GCaMP fluorescence in MBON-γ1pedc>αβ during odor stimulation (12 s vinegar) upon ATP application in an in vivo preparation. Right: Boxplots displaying normalized (to genetic control) responses (area under the curve) to 12 s vinegar and ATP stimulation in MBON-γ1pedc>αβ/MVP2. (G) Epistasis experiment for VPM4 and MBON-γ1pedc>αβ suggesting that VPM4 suppresses MBON-γ1pedc>αβ induced odor tracking. Running speed at trial 1 and 10. (H) Running speeds over trials during odor stimulation period. For all analyses, statistical notations are as follows: ns, p > 0.05; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. In all panels, error bars denote SEM.

Figure 7

Persistence in Odor Tracking Depends on Dopamine Signaling (A) Scheme of dopaminergic neurons innervating MB lobes. (B) Running speed of hungry flies at trial 1 and 10 (24 h starved) with inactivated output of either PAM DANs (58E02-Gal4>UAS-Shi^ts1) or TH+/PPL DANs (TH-Gal4>UAS-Shi^ts1). (C) Average running speed over 10 trials for hungry flies (24 h starved) with inactivated output of PAM DANs (58E02-Gal4>UAS-Shi^ts1) or TH+ DANs (TH-Gal4>UAS-Shi^ts1) compared to control. The boxplot displays the main group effect. (D) Running speed during trial 1 and 10 of hungry flies (24 h starved) with inactivated output of different subsets of DANs within the TH-Gal4 positive DAN cluster. (E) Average running speed during odor stimulation over 10 trials of hungry flies (24 h starved) with inactivated output of different DAN/TH+ subsets. The boxplot displays the main group effect. (F) Table of TH-Gal4 transgenes in different clusters of DANs. (G) Scheme displaying PPL1-γ1pedc (MP1) DAN innervating the MB. (H) Running speed during trial 1 and 10 of hungry flies (24 h starved) with inactivated output of PPL1-γ1pedc (MP1) compared to controls. (I) Average running speed during odor stimulation over 10 trials of hungry flies (24 h starved) with inactivated output of PPL1-γ1pedc (MP1) compared to controls. The boxplot displays the main group effect. (J) Running speed during trial 1 and 10 of hungry flies (24 h starved) lacking the Dop1R2 receptor gene and heterozygous controls. (K) Average running speed during odor stimulation over 10 trials of hungry flies (24 h starved) without Dop1R2 compared to heterozygous controls. The boxplot displays the main group effect. (L) Running speed during trials 1 and 10 for hungry flies (24 h starved) with Dop1R2 receptor knockdown in αβ Kenyon cells (MB008B>Dop1R2i) compared to controls (pBDPU>Dop1R2i). (M) Average running speeds over 10 trials for hungry flies (24 h starved) with Dop1R2 receptor knockdown in αβ Kenyon cells (MB008B>Dop1R2i) compared to controls (pBDPU>Dop1R2i). The boxplot displays the main group effect. (N) Model of neurons implicated in persistent odor tracking (see main text for details). For all analyses, statistical notations are as follows: ns, p > 0.05; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. In all panels, error bars denote SEM.