Dissection of the Drosophila neuropeptide F circuit using a high-throughput two-choice assay

Abstract

In their classic experiments, Olds and Milner showed that rats learn to lever press to receive an electric stimulus in specific brain regions. This led to the identification of mammalian reward centers. Our interest in defining the neuronal substrates of reward perception in the fruit fly Drosophila melanogaster prompted us to develop a simpler experimental approach wherein flies could implement behavior that induces self-stimulation of specific neurons in their brains. The high-throughput assay employs optogenetic activation of neurons when the fly occupies a specific area of a behavioral chamber, and the flies' preferential occupation of this area reflects their choosing to experience optogenetic stimulation. Flies in which neuropeptide F (NPF) neurons are activated display preference for the illuminated side of the chamber. We show that optogenetic activation of NPF neuron is rewarding in olfactory conditioning experiments and that the preference for NPF neuron activation is dependent on NPF signaling. Finally, we identify a small subset of NPF-expressing neurons located in the dorsomedial posterior brain that are sufficient to elicit preference in our assay. This assay provides the means for carrying out unbiased screens to map reward neurons in flies.

Keywords: Drosophila; high-throughput two-choice assay; neuropeptide F; optogenetics; reward circuit.

Fig. 1.

Flies exhibit preference for NPF neuron activation. (A) Schematic representation of the two-choice assay system. Flies are exposed to 617-nm light on only one side of the chambers. (B) Experimental data expressed as the mean ± SE of the preference index (PI) over time. The yellow shading indicates the side and period of activation. The gray boxes represent the periods of time used to calculate the activation effect (AE = mean PI during last minute of activation − mean PI during last minute of acclimation) (n = 13–16). (C) Activation effect for the data in B showing that experimental NPF>CsChrimson flies have a significant preference for activation of NPF neurons compared with control NPF-GAL4 and UAS-CsChrimson flies (n = 13–16; one-way ANOVA with Tukey’s post hoc test; ***P < 0.001) (frequency of activation, 40 Hz; 617-nm LED light intensity, 5 µW/mm²).

Fig. S1.

Frequency dose–response curve for activation of NPF neurons. (A) During each light pulse, the 617-nm LEDs remained ON for 8 ms followed by a variable amount of time, depending on the desired frequency, in which the 617-nm LEDs remained OFF. (B) Activation of NPF neurons at different frequencies, using a 617-nm LED light intensity of 5 µW/mm², reveals that a minimum of 12.5 Hz is required for the flies to display a significant preference (unpaired t test for each frequency between the inactive control group and the experimental group; n = 7–16; **P < 0.01; ***P < 0.001).

Fig. S2.

Flies avoid activation of Gr66a neurons. (A) Experimental data expressed as the mean ± SE of the preference index over time (n = 14). (B) The activation effect for the data in A shows that flies display a significant avoidance for the activation of Gr66a neurons (n = 14; one-way ANOVA with Tukey’s post hoc test; ***P < 0.001) (frequency of activation, 40 Hz; 617-nm LED light intensity, 5 µW/mm²).

Fig. 2.

Flies display preference for NPF neuron activation independently of social (group) context. (A) Traces over time for the proportion of flies on the active side for single experimental NPF>CsChrimson flies (n = 203; Top) or single control UAS-CsChrimson flies (n = 198; Bottom). The yellow box indicates the period of activation. Data are expressed as mean ± SE (frequency of activation, 40 Hz; 617-nm LED light intensity, 5 µW/mm²). (B) ΔTime (%) (preference) of flies during the activation and recovery phases, showing that single flies have a significant preference for the activation of NPF neurons (unpaired t test; ***P < 0.001). (C) Representative traces of experimental (Top) and control (Bottom) flies. Yellow indicates time and side of illumination. (D) Return amplitude data showing that, during the period of activation, experimental flies move for shorter distances into the unilluminated side compared with control flies (unpaired t test; ***P < 0.001).

Fig. 3.

Effect of NPF neuron activation on single fly locomotion. (A and B) Experimental data for NPF>CsChrimson flies (n = 203) (A) or control UAS-CsChrimson flies (n = 198) (B), expressed as the mean ± SE of the speed over time. (C and D) Scatterplot of the ΔTime (%) (preference) vs. speed during activation for single experimental flies (n = 203) (C) or single control flies (n = 198) (D), showing that preference and speed during activation are negatively correlated in NPF>CsChrimson flies (r = −0.332; P < 0.001), but not in control flies (r = 0.017; P > 0.05).

Fig. S3.

Representative traces of single NPF>CsChrimson flies. Representative position over time traces for NPF>CsChrimson flies showing different levels of locomotion during the activation period. Locomotor activity during the acclimation and recovery phases are comparable. The yellow box indicates the side and period of activation (617-nm LED light intensity, 5 µW/mm²; frequency of activation, 40 Hz).

Fig. S4.

Scatterplot of the preference (activation effect) vs. speed during activation for grouped NPF>CsChrimson flies (n = 186) (A) or grouped control flies (n = 179) (B), showing that preference and speed during activation are negatively correlated in NPF>CsChrimson flies (r = −0.2517; P < 0.001), but not in control flies (r = 0.0637; P > 0.05).

Fig. S5.

Scatterplot of the speed during the recovery phase vs. speed during acclimation phase for single NPF>CsChrimson flies (n = 203) (A) or single control flies (n = 198) (B), showing that speed during recovery and acclimation are positively correlated in both NPF>CsChrimson flies (r = 0.7606; P < 0.001) and control flies (r = 0.5813; P < 0.001).

Fig. 4.

Competition between natural and artificial rewards. (A) The chambers were modified by introducing an acrylic divider. A single fly (male or virgin female), expressing CsChrimson in NPF neurons or Gr66a neurons, was introduced into each side of the chamber for 2 min, after which the divider was removed and flies had the possibility to interact with each other for 3 min (flies that either copulated or failed to interact during this period were excluded from the analysis). This was followed by light stimulation (yellow area) in only one side of the chambers for a period of 5 min. (B) Activation effect [ΔTime (%)] data for single NPF>CsChrimson male flies paired with a single male (control group) or single virgin female (experimental group) Gr66a>CsChrimson fly. When paired with a virgin female, male flies show reduced preference for the activation of NPF neurons (n = 21–25; unpaired t test, **P < 0.01). (C) Activation effect [ΔTime (%)] data for flies expressing CsChrimson in Gr66a neurons, for both control and experimental groups. In both cases, flies avoid the activation of Gr66a neurons (n = 21–25; unpaired t test, **P < 0.01) with no difference between them (unpaired t test, P > 0.05) (frequency of activation, 10 Hz; 617-nm LED light intensity, 5 µW/mm²).

Fig. 5.

Optogenetic activation of NPF neurons is rewarding. (A) Flies were trained to associate an odor with the optogenetic activation of NPF neurons in a single training session consisting of 5 min of exposure to odor 1 [ethyl acetate (EA)] coupled with optogenetic activation of NPF neurons [activation at constant light; 617-nm LED light intensity, 20 µW/mm², as described before (29)], followed by 5 min of exposure to air, followed by 5 min of exposure to odor 2 [isoamyl alcohol (IAA)]. To exclude any inherent bias for the olfactory cues, another group was trained in reciprocal manner (group 2). Conditioned odor preference was tested 5 min after the end of the training. Conditioned preference index (CPI) is the average between the CPIs of group 1 and group 2. (B) NPF>CsChrimson flies showed a significant conditioned odor preference (n = 6–8; unpaired t test; ***P < 0.001).

Fig. S6.

Optogenetic activation of Gr66a neurons is aversive. (A) Flies were trained to associate an odor with the optogenetic activation of Gr66a neurons using a single training session consisting of 10-min exposure to air, followed by a 10-min exposure to odor 1 [ethyl acetate (EA)], followed by a 10-min exposure to air, and finally a 10-min exposure to odor 2 [isoamyl alcohol (IAA)] paired with Gr66a neuron activation. To exclude any inherent bias for the olfactory cues, a second group of flies was trained to associate the activation of Gr66a neurons with the reciprocal odor (group 2). Conditioned odor preference was tested 10 min after the end of the training. Each conditioned preference index (CPI) is calculated as the average between the CPIs of group 1 and group 2. (B) Gr66a>CsChrimson flies showed a significant conditioned odor aversion (n = 12; unpaired t test; **P < 0.01) when the memory was tested 10 min after training (frequency of activation, 40 Hz; 617-nm LED light intensity, 5 µW/mm²).

Fig. 6.

NPF is required for the NPF activation-induced preference. Distribution of NPF neurons in the central brain (A) and ventral nerve cord (B) of the fly, as visualized by the expression of the cell polarity markers DenMark (dendrites) and Syt::GFP (synaptic terminals) (42, 43) driven by the NPF-GAL4 driver. Positions of the large P1 and L1-l neurons, and the FSB are indicated. (C) Flies, in which the expression of NPF was targeted using an RNAi construct, showed a reduced preference for the activation of NPF neurons (n = 8–12; unpaired t test, *P < 0.05; **P < 0.01) (617-nm LED light intensity, 5 µW/mm²). (Magnification: 20×.)

Fig. S7.

Presence of extra UAS binding sites does not change the preference for NPF neuron activation. Positive control NPF>CsChrimson flies (left bar) carry NPF-GAL4 and UAS-CsChrimson; experimental flies carry, in addition, 20XUAS-mCD8-GFP (n = 10–12; unpaired t test, P > 0.05) (frequency of activation, 40 Hz; 617-nm LED light intensity, 5 µW/mm²).

Fig. 7.

Expression of NPF and NPF-GAL4. (A) The NPF-GAL4 expression pattern includes two large neurons (P1 and L1-l) per hemisphere. (B) Higher-magnification view of the area indicated by the dashed box in A, showing that NPF is also expressed in several small neurons, located in the dorsal medial brain (DM) (left-facing arrowhead). Images correspond to the second third of the confocal stack. (C) Same area as shown in B, but images correspond to the first third of the confocal stack, which allows for the visualization of the cell bodies of the small neurons projecting to the FSB (horizontal arrows). Images correspond to the maximum intensity projection of different portions of a confocal stack collected from the posterior to the anterior end of the brain. Green: NPF-GAL4 expression pattern (i). Magenta: endogenous NPF expression (ii). White in iii: overlapping of NPF-GAL4 expression and NPF endogenous expression. (Magnification: 20×.)

Fig. S8.

Neuroanatomy of neurons expressing NPF-GAL4. Representative multicolor flip-out (MCFO) results, showing the different types of NPF-GAL4 neurons: large dorsal medial (P1) neuron (A); large dorsal lateral (L1-l) neuron (A and B); small fan-shaped body (P2) interneurons (B and C); small dorsal medial (DM) neuron. (D) Schematic representation of the different subtypes of neurons expressing NPF-GAL4. In all images, small arrows indicate the projections from P2 neurons to the fan-shaped body, while big arrowheads indicate the neuron of interest in each panel. The male-specific neurons L1-s, D1, and D2 (20) were rarely labeled and have therefore been omitted. (Magnification: 20×.)

Fig. 8.

Preference for the activation of specific subsets of NPF neurons. (A) Intersection of the NPF-LexA and NPFR-GAL4 drivers, yielding expression in only the big (L1-l + P1) NPF neurons. (B) The ss0020 split-GAL4 line labels a subset of P2 NPF neurons. Brains were imaged from posterior to anterior. Magenta: endogenous NPF. Green: CsChrimson::mVenus. (C) Activation of neither the big (L1-l + P1) nor P2 NPF neurons is sufficient to recapitulate the effect of activating all NPF neurons [n = 14–15; one-way ANOVA followed by Tukey's test. **P < 0.01] (frequency of activation, 40 Hz; 617-nm LED light intensity, 20 μW/mm²). (Magnification: 20×.)

Fig. 9.

DM NPF neurons are sufficient to induce preference in the two-choice arena. (A and B) Representative brains of single flies expressing CsChrimson in DM neurons only (A), or in DM plus other neurons (B) (example shows expression in two neurons projecting to the FSB, arrows). (C) Experimental data expressed as the mean ± SE of the proportion of flies on the active side over time. The yellow box indicates the period of activation (DM neurons only, n = 19; DM plus other neurons, n = 19). (D) ΔTime (%) (preference) of flies during the activation phase, indicating no significant difference between the preference of flies with CsChrimson expression in DM neurons only or in DM plus other neurons. Female flies were used in this experiment as stochastic labeling was sparser than in males (unpaired t test; P > 0.05) (frequency of activation, 40 Hz; 617-nm LED light intensity, 5 µW/mm²). (Magnification: 40×.)

Fig. S9.

Representative examples of flies that showed expression of CsChrimson in a subset of DM NPF neurons (A and B), which also showed a clear preference response during the assay (C and D). Green: CsChrimson::mVenus. (Magnification: 20×.)