Satiation state-dependent dopaminergic control of foraging in Drosophila

Hunger evokes stereotypic behaviors that favor the discovery of nutrients. The neural pathways that coordinate internal and external cues to motivate foraging behaviors are only partly known. Drosophila that are food deprived increase locomotor activity, are more efficient in locating a discrete source of nutrition, and are willing to overcome adversity to obtain food. We developed a simple open field assay that allows flies to freely perform multiple steps of the foraging sequence, and we show that two distinct dopaminergic neural circuits regulate measures of foraging behaviors. One group, the PAM neurons, functions in food deprived flies while the other functions in well fed flies, and both promote foraging. These satiation state-dependent circuits converge on dopamine D1 receptor-expressing Kenyon cells of the mushroom body, where neural activity promotes foraging independent of satiation state. These findings provide evidence for active foraging in well-fed flies that is separable from hunger-driven foraging.


Sensory and Nutritional Inputs to Food Seeking.
We tested for the role of olfaction, taste, and vision in foraging behavior in food-deprived flies ( Fig. 2A). Neither genetic nor surgical ablation of food odor-detecting neurons -olfactory coreceptor mutant Orco 1 or removal of the third antennal segment -affected food occupancy 24,25 . Similarly, flies lacking a subset of sugar sensing taste receptors showed normal food occupancy for sucrose. These experiments suggested that flies may use more than one sensory modality when seeking nearby food. Flies with both ablated antennae and taste receptor mutations showed decreased food occupancy, suggesting coordination between olfaction and taste. Food occupancy remained robust in complete darkness. However, taste receptor mutant flies showed reduced food occupancy in total darkness, and additionally removing olfactory input did not further reduce occupancy. These results indicate that flies use a combination of taste, olfactory, and visual cues to find and occupy a discrete food source.
Flies may seek one or more food constituents. Food deprived flies were most attracted to complete food, then sugars, and then protein (Fig. 2B). In a binary choice competition where flies are presented with two closely apposed sources, flies preferred complete food over any other option, and sugars over yeast ( Supplementary  Fig. S1). Similarly, flies preferred nutritious and sweet sucrose more than sweet-only sucralose ( Supplementary  Fig. S1). Finally, nutrition appears to be important for switching the locomotor state of food deprived flies: when given a single source, flies slowed more in the presence of sucrose or D-glucose, compared to sweet only sucralose or L-glucose, respectively (Fig. S1D,E). These findings suggest that sweetness is a mechanism that captures flies on a food source, and that nutritional content is important for fully switching flies from the pre-ingestive to consummatory phase of foraging.
A characteristic of motivated behavior is the willingness to overcome negative consequences 4 . Flies will eat substantially less food when it is adulterated with bitter compounds, and this scales with satiation state 13,26 . In a binary choice competition, food deprived flies occupied quinine-containing food, but only if there was no better choice (Fig. 2C). Furthermore, food intake under one-choice conditions was less suppressed by quinine with a longer period of deprivation (Fig. 2D). We used a sucrose food source for all subsequent experiments.

Role of Dopaminergic Neurons in Food
Seeking. Dopaminergic neural circuits are critical for motivation, reward, and foraging in mammals, and for many similar functions in flies 27 . To test the role of dopamine in foraging in flies, we acutely inactivated and activated subsets of dopamine neurons in fed and food-deprived flies and assessed occupancy of sucrose. Dopamine neurons group into several discrete anatomical and functional clusters in the adult fly brain (Fig. 3C). TH-Gal4 labels most dopamine neuron clusters, but is largely absent from the PAM (protocerebral anterior medial) cluster of approximately 130 dopamine neurons 28 . 0273-Gal4 labels most dopamine neurons in the PAM cluster but not other dopamine neurons 29 . Acutely blocking transmitter release in TH-Gal4 neurons with the temperature-sensitive dynamin Shibire (Shi ts ) had no effect on food occupancy in food deprived animals (Fig. 3A). Food occupancy was decreased when TH-Gal4 neurons were transiently inactivated in fed animals. There was no effect of inactivation on locomotor activity ( Supplementary Fig. S2). Conversely, inactivation of 0273-Gal4 neurons decreased food occupancy in food deprived but not fed animals. DAT-Gal80 (R58E02-Gal80) expresses the GAL4 inhibitor GAL80 exclusively in PAM neurons: DAT-Gal80 blocked the 0273 > Shi ts food occupancy phenotype (Fig. 3A) 17 . Finally, chemical depletion of dopamine with 3-iodotyrosine also decreased food occupancy, indicating that dopamine is a neurotransmitter for foraging ( Supplementary  Fig. S2). Thus, dopamine neurons in the TH-Gal4 pattern promote food occupancy in fed animals, and PAM dopamine neurons in the 0273-Gal4 pattern promote food occupancy in food deprived animals.  To test if dopamine neurons are permissive or instructive, we acutely activated them using the temperature-sensitive cation channel TrpA1. Consistent with an instructive role, activating TH-Gal4 neurons in fed flies increased food occupancy (Fig. 3B). Fed 0273 > TrpA1 flies showed a marked decrease in food occupancy, and this was due to PAM dopaminergic activation in the 0273-Gal4 pattern.
To identify the relevant neurons in the TH-Gal4 pattern, we used transgenes that differentially label specific clusters of dopamine neurons (Fig. 3H) 17 . Activation of TH-C' that included the PPL2ab, PPM2, and PAL, but not the PPL1, PPM1, or PPM3 dopamine neuron clusters increased food occupancy in fed flies (Fig. 3D). Conversely, inactivation of TH-D' that includes PPL1, PPM2, and PPM3 neurons decreased food seeking in fed flies (Fig. 3E). The PPL1 neurons are particularly well-characterized for their roles in both appetitive and aversive learning and memory. Inactivation of neurons in the NP2758 pattern that includes PPL1-γ1pedc (MB-MP1) PPL1 and no other dopamine neurons decreased food occupancy in the fed state (Fig. 3F). To test if the identified dopaminergic neurons may regulate feeding motivation, we activated TH-Gal4 neurons in mildly (4 hr) food-deprived flies. Under these conditions, activation of TH-Gal4 neurons specifically increased consumption of quinine adulterated food (Fig. 3E).
Taken together, these experiments are consistent with dual roles for dopamine in foraging behavior: a PAM dopamine neuron-mediated promotion in the food-deprived state, and a TH-Gal4 dopamine neuron-mediated promotion in the fed state. PPL1-γ1pedc neurons in the TH-D' pattern are necessary, and distinct neurons in the TH-C' pattern are sufficient for promoting food occupancy in the fed state. PAM dopamine neurons can block foraging in the fed state.
Dopamine Receptor Regulation of Food Seeking. Dop1R1 encodes a D1-like dopamine receptor that functions in motivation-related behaviors, including arousal state, drug reward, and learning and memory [30][31][32] . We tested flies with strongly reduced expression of Dop1R1 for foraging behaviors. Food-deprived Dop1R1 mutant flies were hyperactive and appeared to ignore food (Fig. 4A). Moreover, Dop1R1 mutant food occupancy was reduced when fed or food deprived (Fig. 4B). Loss of the dopamine D2-like receptor D2R did not affect food occupancy, but did restore normal food occupancy to Dop1R1 mutants. The simplest explanation is that Dop1R1 promotes foraging, and that an opposite role for D2R is uncovered in the absence of Dop1R1. Food intake was unaffected in food-deprived flies of these genotypes ( Supplementary Fig. S3).

The Mushroom Bodies Promote Food Seeking Independent of Satiation State. We performed
genetic rescue experiments to ask where Dop1R1 functions for foraging in food deprived flies. To bias the rescue towards functionally relevant brain regions, we utilized Dop1R1-Gal4 strains that expressed GAL4 under the control of short non-coding genomic DNA fragments cloned from the Dop1R1 locus (Fig. 4C) 33 . Food occupancy was partially rescued when Dop1R1 was expressed with three different Dop1R1-Gal4 strains in food-deprived Dop1R1 mutants: B07, B12, and C02 (Fig. 4D). Anatomical analysis of the expression patterns for the rescuing Dop1R1-Gal4 drivers revealed expression overlap. In the B12 and C02 strains, the mushroom bodies were prominently labeled, as were regions of the central complex, including the fan-shaped body and protocerebral bridge (Fig. 4F,G). The B07 strain prominently labeled the ellipsoid body of the central complex (Fig. 4H). We failed to rescue Dop1R1 mutant food occupancy using GAL4 drivers that label the ellipsoid body, fan-shaped body, or the protocerebral bridge (not shown). By contrast, decreasing GAL4 activity with mushroom body-specific expression of GAL80 (MB247-Gal80) eliminated B12 rescue of the Dop1R1 mutant food occupancy phenotypes (Fig. 4E) 34 . Moreover, restoring Dop1R1 with the mushroom body-specific driver MB247-Gal4 rescued Dop1R1 food occupancy (Supplementary Fig. S3). Thus, Dop1R1 expression in the mushroom bodies is sufficient to promote foraging in food deprived animals.
We next tested the role of neurotransmission in Dop1R1-expressing mushroom body neurons. Similar to loss of Dop1R1, acute blockade of synaptic output in B12 neurons with Shi ts decreased food occupancy in both fed and food-deprived flies (Fig. 4I). Importantly, this effect also localized to the mushroom bodies (Fig. 4J). B12 > Shi ts flies also showed reduced locomotion, however this phenotype persisted when the mushroom body neurons were subtracted from B12 ( Supplementary Fig. S3), suggesting that distinct Dop1R1 neurons control food occupancy and locomotion. Finally, acute activation of B12 neurons in fed flies increased food occupancy (Fig. 4K). Taken together, these results indicate that the activity of Dop1R1-expressing mushroom body neurons promote foraging in both the fed and food-deprived state.

Discussion
Distinct dopaminergic circuitry promotes foraging under well fed and food deprived conditions. Dopamine neurons in the TH-C′ pattern promote foraging in well fed flies, and dopamine neurons in the PAM cluster promote foraging in food deprived flies. The PAM neurons likely function in a direct circuit with Dop1R1-expressing Kenyon cell neurons of the mushroom body that promote foraging in both the fed and food-deprived states. These circuits function under conditions where flies can freely perform many steps of foraging behavior. Understanding how these dopaminergic circuits contribute to discrete steps of feeding behavior, from local search through to repletion and disengagement from a food source, will help define how motivational states transition from task to task.

Roles of Dopamine in Appetitive Behaviors. Dopaminergic neurons are critical for many appetitive
and aversive behavioral responses across animal species. Dopamine may act as a salience, arousal, or attention signal that gives importance to specific valence information arriving from other circuit elements 27,35,36 . In rodents, genetic, pharmacological, and lesioning studies indicate that striatal dopaminergic pathways can selectively function in the pre-ingestive phase to promote food seeking 35,37,38 . We found that acute activation of dopamine neurons in fed flies increased food occupancy, yet it did not cause increased food intake. Likewise, genetic elimination  of the Dop1R1 receptor decreased food occupancy without affecting food intake. In contrast, inactivation of Dop1R1 receptor neurons decreased food intake in the food-deprived state, possibly reflecting their broader role in integrating sensory and internal state information (not shown). These findings suggest that dopaminergic pathways promote pre-ingestive food seeking. However, the role of dopamine is more complex. For example, the PAM dopamine neurons are activated by ingestion of sugar, and their activation is greater in food-deprived flies, indicating that dopaminergic neurons are engaged during the consummatory phase of feeding, and they may be sensitized to responding to input during the pre-ingestive phase 17 . Furthermore, specific dopamine neurons respond to other food-relevant environmental cues such as protein and water 14,39,40 .
Prior studies assigned dopamine to particular aspects of feeding behavior and also to motor functions that are critical to foraging 14,23 . In particular, dopamine neurons in the TH-Gal4 pattern are implicated in controlling motor output: TH-Gal4 neuron hyperpolarization, blocking synaptic input, interferes with motor performance and aspects of foraging behavior in food deprived flies 23,28 . We did not detect differences in unstimulated motor activity or in the magnitude of an olfactory-stimulated startle response when we blocked synaptic output from TH-Gal4 neurons, indicating that flies exhibited grossly normal motor behavior in our assay 41 . The differences in observed phenotypes may reflect the multifunctional roles of TH-Gal4 dopamine neurons that are revealed by specific types of manipulation.
Which dopamine neurons are responsible for foraging? In well-fed flies, neurons in the TH-Gal4 pattern are both necessary and sufficient to promote foraging. TH driver transgenes that express in a more restricted pattern allowed us to separate these roles. TH-C' neurons are sufficient, but not necessary, to promote foraging. This pattern includes dopamine neurons in the PAL, PPM2, and PPL2 clusters. TH-C' neurons were previously shown to promote protein consumption and, separately, egg-laying preference on sucrose 17,42 . Individual neurons in the PPM2 cluster, the DA-WED neurons, support protein consumption preference in protein deprived flies 14 . The DA-WED neurons synapse to Dop1R1 neurons in the B03 pattern, which did not support rescue of food seeking in our experiments. However, the B03 rescue was, by necessity, done in food deprived flies, when TH neurons were dispensable for foraging. Thus, it is possible that protein consumption preference and foraging are encoded by the same dopaminergic circuit that is used under different nutritional states and goals. Separately, dopamine neurons in the TH-D' pattern are necessary, but not sufficient, to promote foraging. Inactivation of the PPL1-γ1pedc (MB-MP1) PPL1 neurons (using NP2758-Gal4), also decreased food occupancy, suggesting that these dopamine neurons are permissive for foraging in fed flies 16 . The PPL1-γ1pedc neurons are implicated in the formation of aversive memories in well-fed flies, and their activity is downregulated by food deprivation [43][44][45][46][47] . Our findings argue that there are distinct dopaminergic circuits in the TH-Gal4 pattern that control different aspects of food seeking in the well-fed state. The PAM neurons are also heterogeneous, sending projections that tile to well-defined regions of the mushroom body and to regions of the protocerebrum. Specific subsets of PAM neurons that are included in the 0273-Gal4 pattern have been implicated in various forms of appetitive learning and memory, however inactivation of these more specific PAM neuron subsets did not impact food seeking in food deprived flies (not shown) 15,17,[48][49][50] . This suggests that there may be further segregation of PAM dopamine neuron function, possibly according to innate and learned appetitive responses.

Sensory Tuning of Food Seeking Motivation.
Appetitive olfactory cues such as those emitted from palatable food elicit approach and can activate neurons important for feeding 51,52 . Olfactory receptor neurons that respond to appetitive odors increase sensitivity through the actions of the neuropeptides sNPF and SIFamide 21,53 . Further, neurons that release the neuropeptide NPF are activated to a greater extent in response to food odors in food-deprived flies; their activation promotes and inactivation inhibits odor attraction 51 . In well-fed larvae, the attractive odor pentyl acetate increases food intake through the actions of NPF and dopamine 11 . Therefore, food-related odors not only elicit approach behavior in a satiation state dependent manner, but also increase the activity of neurons expressing neuropeptides that regulate feeding behavior. Our results indicate that olfaction is important but apparently not crucial for food seeking in food-deprived flies: neither surgical nor genetic ablation of olfaction decreased food occupancy, and its role was only revealed by simultaneous partial ablation of taste responses. Further, flies were efficient in seeking odorless sucrose. Taken together, olfaction, hygrosensation, visual cues, and taste responses likely act in concert with internal cues to set the intensity of foraging when freely behaving flies are in close proximity to a food source.
Behavioral Measurements. Groups of 21 males were collected 1-2 days prior to the experiment. A group is an n = 1. For food deprivation, flies were placed into empty culture vials containing water saturated Whatman filter paper. For 3-iodotyrosine treatment, flies were cultured for 30 hr with 5% sucrose/2% yeast/10 mg/mL 3-iodotyrosine (3IY), and treated an additional 16 hr with 3IY in water for food deprivation. Standard fly food SCientifiC REPORTS | (2018) 8:5777 | DOI:10.1038/s41598-018-24217-1 was used as the food source in the arenas for all experiments except where indicated. Approximately 100 μL of food or 1.25% agarose with additives was pipetted onto a small square of Parafilm and kept humidified. Thin-walled Plexiglas behavioral chambers were designed with two side-by-side arenas, each arena measuring 45 × 75 × 10 mm, or 85 × 135 × 10 mm for experiments with Shibire ts . Chambers were designed and built by IO Rodeo; design files are available (Pasadena, CA). Chambers, food sources, and flies were acclimated to the testing temperature prior to introducing them into the behavioral arena. A Pelltier incubator was used for experiments performed at lowered and elevated temperatures (IN45, Torrey Pines Scientific). Flies were filmed from above at 10 fps with the arena placed on white light LED panel (Edmund Optics). Filmed flies were tracked with customized DIAS software as previously described 54 . For food occupancy, the number of flies off food was subtracted from the total number of flies and divided by total number of flies. In binary choice experiments, the food sources were deposited in direct apposition and placed at the center of the arena, and the number of flies on each source was manually counted. Percent on food was calculated as the average of the last two measured time points (20-30 min). Locomotor activity was the average speed of all flies in 20 sec bins measured for a 1 min interval at 20 and 30 min.
To measure food intake, 5 ml standard fly food with 2% erioglaucine (Sigma) with or without 3 mM quinine was striped onto 1/4 of the inner surface of a wide fly vial, and condensation removed. 30-50 flies were introduced and the vial laid on its side so that the food edge was at the apex. After 30 min, the flies were homogenized in a volume adjusted to the number of flies and consumption was determined spectrophotometrically.
Statistical measurements were made with Prism 6.0 (GraphPad). One-way ANOVA followed by Tukey's post-hoc comparisons (or Bonferroni post-hoc planned comparison) were used when data did not show unequal variance by the Brown-Forsythe test, otherwise the Kruskal-Wallis test followed with Dunn's post-hoc was used. t-tests were two-tailed. Error bars are the SEM. Data is available upon request.