Article

A Circuit that Regulates Ingestion by Integrating Food and Hunger Signals

Graphical Abstract Authors Nilay Yapici, Raphael Cohn, Christian Schusterreiter, Vanessa Ruta, Leslie B. Vosshall

Correspondence [email protected]

In Brief A neural circuit that connects sweet taste in the with local in the primary taste center allows flies to regulate their ingestion of food by integrating information about hunger state and food quality.

Highlights d Expresso system measures single fly ingestion in real time at nanoliter resolution d Flies regulate ingestion by integrating hunger state and food quality d IN1 interneurons receive input from pharyngeal taste neurons and regulate ingestion d IN1 neurons respond to sucrose ingestion in a hunger-state- dependent manner

Yapici et al., 2016, Cell 165, 715–729 April 21, 2016 ª2016 Elsevier Inc. http://dx.doi.org/10.1016/j.cell.2016.02.061 Article

A Taste Circuit that Regulates Ingestion by Integrating Food and Hunger Signals

Nilay Yapici,1 Raphael Cohn,2 Christian Schusterreiter,3,4 Vanessa Ruta,2,5 and Leslie B. Vosshall1,5,6,* 1Laboratory of Neurogenetics and Behavior, The Rockefeller University, New York, NY 10065, USA 2Laboratory of Neurophysiology and Behavior, The Rockefeller University, New York, NY 10065, USA 3Department of Computer Science, University of Oxford, Oxford OX1 3QD, UK 4Ticomo Research GmbH, 6300 Zug, Switzerland 5Kavli Neural Systems Institute, New York, NY 10065, USA 6Howard Hughes Medical Institute, New York, NY 10065, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2016.02.061

SUMMARY nutrient sensing to regulate subsequent eating behavior, which may be too slow to control the amount of food ingested (Dus Ingestion is a highly regulated behavior that inte- et al., 2015; Miyamoto et al., 2012; Zukerman et al., 2011). The grates taste and hunger cues to balance food intake biology of ingestion is poorly understood. with metabolic needs. To study the dynamics of Mechanisms of peripheral taste processing and the regulation ingestion in the vinegar fly Drosophila melanogaster, of the hunger state have been studied intensively in vertebrates. we developed Expresso, an automated feeding Taste receptors in the mouth and cortical regions in the brain that assay that measures individual meal-bouts with respond to taste qualities have been identified (de Araujo and Simon, 2009; Barretto et al., 2015; Chandrashekar et al., 2010; high temporal resolution at nanoliter scale. Flies Chen et al., 2011; Huang et al., 2006; Mueller et al., 2005; Nelson showed discrete, temporally precise ingestion that et al., 2001; Zhao et al., 2003). The activation of sweet cells was regulated by hunger state and sucrose concen- promotes food acceptance in hungry animals, while the activa- tration. We identify 12 cholinergic local interneu- tion of bitter cells stimulates food avoidance (Mueller et al., rons (IN1, for ‘‘ingestion neurons’’) necessary for 2005; Zhao et al., 2003). Neurons in the hypothalamic neuroen- this behavior. Sucrose ingestion caused a rapid and docrine circuits express proopiomelanocortin (POMC), agouti- persistent increase in IN1 activity in related peptide (AgRP), and melanocortin receptor (MC4R) and fasted flies that decreased proportionally in response orchestrate ingestion in response to the hunger state of the ani- to subsequent feeding bouts. Sucrose responses of mal (Aponte et al., 2011; Atasoy et al., 2012; Carter et al., 2013; IN1 interneurons in fed flies were significantly smaller Fan et al., 1997). and lacked persistent activity. We propose that The mechanisms controlling taste and food intake in insects are remarkably similar to those in vertebrates. Drosophila flies IN1 neurons monitor ingestion by connecting sugar- detect and evaluate food using taste cells located in the pe- sensitive taste neurons in the pharynx to neural riphery (Stocker, 1994). The insect equivalent of the vertebrate circuits that control the drive to ingest. Similar mech- is the labellum on the proboscis. This structure is deco- anisms for monitoring and regulating ingestion may rated with taste sensilla that house gustatory neurons, which exist in vertebrates. express gustatory receptors (GRs) that respond to sweet or bitter tastants (Chyb et al., 2003; Clyne et al., 2000; Dahanukar INTRODUCTION et al., 2001, 2007; Scott et al., 2001; Weiss et al., 2011). Stimula- tion of sweet taste neurons in the labellum and legs triggers All animals must simultaneously integrate external sensory stim- extension of the proboscis in fasted flies, followed by initiation uli with internal state to control behavioral decisions (Davis, of food intake (Dethier, 1976; Dethier et al., 1956). Upon inges- 1979; Tinbergen, 1951). One behavior under strict neural and tion, food comes in contact with taste neurons located in the metabolic control is eating, which is regulated both by peripheral pharynx (Stocker, 1994). The function of these pharyngeal taste sensory detection of food and by internally generated states of neurons is poorly understood, but a subset has been shown to hunger and satiety (Brobeck et al., 1943; Burton et al., 1976; regulate sugar ingestion (LeDue et al., 2015). Hoebel and Teitelbaum, 1962; Kennedy, 1953; Mayer, 1953; Taste afferents from the mouthparts and pharynx Raubenheimer and Simpson, 1997; Read et al., 1994). Perturba- target distinct regions of the subesophageal zone (Ito et al., tions in these homeostatic systems can lead to obesity and 2014), the taste center of the fly brain (Marella et al., 2006). associated health problems (Morton et al., 2014). In both verte- This is a densely innervated brain structure housing projection brates and insects, the optimization of food intake requires tight neurons, interneurons, and motor neurons that are required regulation of behaviors responsive to food quality and hunger for taste acceptance, along with motor circuits that regulate state. Once food is ingested, it takes several minutes for enteric ingestion (Flood et al., 2013; Gordon and Scott, 2009; Kain and

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716 Cell 165, 715–729, April 21, 2016 Dahanukar, 2015; Manzo et al., 2012; Marella et al., 2012; assay to quantify the interactions of a fly with a food source Miyazaki et al., 2015; Pool et al., 2014). Additionally, neuromo- (Itskov et al., 2014; Ro et al., 2014). These assays provide a proxy dulators, including dopamine, , neuropeptide F, and for appetitive behavior, but they do not directly measure food short-neuropeptide F, modulate food intake by altering the ac- ingestion. The capillary feeder (CAFE) assay (Ja et al., 2007) mea- tivity of sensory neurons that detect food stimuli or by changing sures ingestion directly from groups of flies, and the the manual- the activity of homeostatic neurons that regulate hunger (Albin feeding (MAFE) assay measures ingestion bouts of individual et al., 2015; Inagaki et al., 2012, 2014b; Root et al., 2011). flies (Qi et al., 2015). Because both require manual measure- We hypothesized that there must be a rapid sensor of inges- ments, their temporal resolution and/or throughput are limited. tion interposed between peripheral taste detection and meta- To investigate factors regulating the temporal dynamics of bolic nutrient sensing. To identify such a sensor in Drosophila, food ingestion, we developed an automated version of the we first developed a high-resolution food ingestion assay, CAFE assay called ‘‘Expresso,’’ which allows us to capture Expresso. This system measures ingestion of individual flies in nanoliter-volume meal-bouts of single flies simultaneously, real time at nanoliter resolution. We used Expresso to show with high temporal resolution. Expresso consists of multiple that flies make rapid feeding decisions based on hunger state single-fly feeding chambers, each connected to a sensor bank and sucrose concentration. We identify 12 cholinergic local inter- (Figures 1A and 1B; Figures S1A–S1E). When a fly drinks from neurons (IN1, for ‘‘ingestion neurons’’) in the taste center of the the capillary, Expresso analysis software automatically detects fly brain that regulate sucrose ingestion. IN1 neurons receive individual meal-bouts in real time by measuring the rapid selective input from sweet taste neurons in the pharynx (LeDue decrease in liquid level (Figure 1C; Figures S1A–S1C; Movie S1). et al., 2015). We used in vivo functional calcium imaging to Accuracy was verified by manually annotating videos of fly show that IN1 activity increased rapidly in hungry flies after ingestion, while ingestion volume was simultaneously monitored sucrose ingestion, switching these neurons into a state of by the Expresso. The concordance between Expresso (hardware sustained activation that lasted for minutes. As the fasted fly and software) and the human observer was 94% for all bouts and continued to ingest sucrose over the course of the experiment, 100% for bouts larger than 10 nl (Figure S1D). Meal-bout volumes the activity of IN1 neurons progressively decreased. Sustained of fasted flies varied between 6 and 200 nl, and volume was highly IN1 activation was strongly attenuated in fed flies offered su- correlated with duration (Figure S1E). We compared the feeding crose or in fasted flies offered a lower concentration of sucrose. behaviors of male and female flies fasted for 24 hr and confirmed Furthermore, when IN1 neurons were optogenetically activated that flies of both sexes consumed liquid food robustly (Ja et al., in fed flies, they ingested food as if they were fasted. Our work 2007)(Figures S1F and S1G). We also quantified several mea- provides functional evidence for the existence of a taste circuit sures that captured the dynamics of food ingestion (Figures that food intake via the pharynx and rapidly integrates S1H–S1N). Female flies showed a small increase in latency but taste information with hunger state to control the rate, volume, consumed food more rapidly than males (Figures S1K and S1L). and timing of ingestion. Fasted flies concentrated their meal-bouts at the beginning of the experiment and rapidly reached half-total ingestion (median, RESULTS 34.77 s in females and 56.44 s in males) (Figures S1M and S1N).

Expresso, a High-Resolution Feeding Assay Temporal Dynamics of Ingestion Are Dramatically No existing fly-feeding assay measures real-time ingestion by Altered by Hunger and Sucrose Concentration single flies at high resolution (Cognigni et al., 2011; Deshpande Hunger state and the quality of food offered affect ingestion et al., 2014; Edgecomb et al., 1994; Navawongse et al., 2015). (Ostlund et al., 2013; Spector et al., 1998). We examined this The proboscis extension assay measures the likelihood that by progressively fasting flies and monitoring sucrose ingestion. a fly will extend its proboscis in response to food (Shiraiwa and Most of the fed flies (0 hr fasted) did not consume 1 M sucrose. Carlson, 2007). Two groups recently automated this manual As fasting time increased, more flies initiated feeding with shorter

Figure 1. Fasting Tunes the Temporal Dynamics of Sucrose Ingestion (A) Expresso photodiode sensor (left) and side view schematic of the Expresso system (right). (B) Front view of the Expresso system (scale bar, 20 mm). (C) Real-time liquid food level measurements of a single 24-hr-fasted male fly offered standard liquid food. Detected meal-bouts are shown in magenta. Note that these data are reprinted in the lower panel in Figure S1C. (D) Meal-bout raster plots of n = 20 male flies fasted for the indicated time before testing. (E) Total ingestion of all flies per 50-s bin over a 33-min experiment. (F) Feeding success (chi-square test, pairwise post hoc comparisons with Fisher’s exact test with Bonferroni correction, p < 0.005; population percentage and 95% confidence intervals). (G and H) Latency to first bout (G), and total sucrose ingestion (H). p < 0.05, Kruskal-Wallis test, pairwise post hoc comparisons using Tukey and Kramer (Nemenyi) tests; n = 20. (I) Gray lines show raw data, and green lines show the nonlinear regression. (J) Bout rate. p < 0.05, Kruskal-Wallis test, pairwise post hoc comparisons using Tukey and Kramer (Nemenyi) tests. (K) Green lines show the nonlinear regression, and gray shades indicate mean ± SEM.

(L) Time to half-total intake (T1/2). p < 0.05, Kruskal-Wallis test, pairwise post hoc comparisons using Tukey and Kramer (Nemenyi) tests; n = 20. Bar plots or box plots labeled with different letters in a given panel are significantly different. See also Figure S1 and Movie S1.

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Figure 2. Fasted Flies Adjust Ingestion Dynamics According to Sucrose Concentration (A) Meal-bout raster plots of 24-hr-fasted male flies (n = 20) offered sucrose at the indicated concentration. (B) Total ingestion of all flies per 50-s bin over a 33-min experiment. (legend continued on next page)

718 Cell 165, 715–729, April 21, 2016 latencies and higher rates, resulting in greater consumption of Using a membrane-tethered GFP, we detected 57F03> in 1 M sucrose (Figures 1D–1H). 24-hr-fasted flies consumed 1 M small numbers of neurons in the brain (Figure 3B), ventral nerve sucrose at much higher rates than fed flies (Figures 1I and 1J). cord (Figure 3C), and taste neurons in the labellum that express These hungry flies concentrated their meal-bouts within the first the chemosensory receptor Ir25a (Benton et al., 2009)(Fig- minute (median half-total ingestion, 11.31–27.74 s) (Figures 1K ure 3D). 57F03> also labeled non-neuronal, non-glial cells in and 1L), and their rate of ingestion decreased rapidly thereafter legs, maxillary palp, and antenna (data not shown). In the brain, (Figures 1I and 1J). 57F03> expression was limited to the subesophageal zone Next, we tested the effects of sucrose concentration on inges- (Figure 3B). tion dynamics. At all sucrose concentrations tested, the number Because the original screen used chronic synaptic silencing, of 24-hr-fasted flies feeding was the same (Figure 2C), but we asked whether acute neuronal silencing also disrupted feeding dynamics were strikingly different (Figures 2A–2J). Flies ingestion. We conditionally inactivated 57F03> neurons with consumed greater volumes of 100 mM sucrose than 1 mM, the temperature-sensitive dominant-negative form of dynamin 10 mM, and 1 M sucrose, indicating that both the taste, caloric (shits). At temperatures above 29C, shits reversibly inhibits syn- content, and amount of sucrose determine the total amount of aptic transmission by blocking membrane recycling (Kitamoto, ingestion at longer timescales (Figures 2D–2F). At lower sucrose 2001). The percentage of 24-hr-fasted 57F03> shits flies that concentrations, flies consumed short meal-bouts spaced over consumed 1 M sucrose at the permissive temperature of 23C the 33-min experiment, and the overall volume consumed was was normal (Figures 3E–3G). However, silencing these neurons low (Figures 2D–2F). As sucrose concentration increased, the in- at the restrictive temperature of 30C caused 57F03> shits flies tensity of ingestion increased, leading to higher bout volumes and to alter the temporal structure of feeding, resulting in a dramatic ingestion rates (Figures 2F–2H). This higher intensity feeding was reduction in total ingestion, bout volume, and ingestion rate also temporally concentrated, with animals that were offered 1 M (Figures 3H–3J). sucrose reaching median half-total ingestion after 18.25 s (Figures 2I and 2J). In contrast, median half-total ingestion at lower sucrose Cholinergic Local Interneurons Control the Dynamics of concentrations ranged from 153 s to 520 s (Figures 2I and 2J). Sucrose Ingestion To clarify which 57F03> neurons are functionally required for A Genetic Screen for Neurons that Regulate Ingestion food ingestion, we inhibited the activity of Gal4 in selected tis- Post-ingestive nutrient-sensing pathways compute the caloric sues and cell types using the Gal80 repressor (Lee and Luo, content of ingested food and regulate subsequent feeding. How- 1999). 57F03> tetanus toxin flies expressing pan-neuronal ever, this response is relatively slow, developing over a period of Elav-Gal80 (Yang et al., 2009) exhibited normal feeding, confirm- minutes (de Araujo et al., 2008; Dus et al., 2011; Miyamoto et al., ing that the 57F03> effect on feeding is due to the silencing of 2012; Zukerman et al., 2011). We hypothesized that flies have a neurons (Figures S2G and S2H). We ruled out a role for taste mechanism to rapidly integrate hunger state with sucrose con- neurons in the labellum, interneurons in the ventral nerve cord, centration to regulate moment-to-moment ingestion. This mech- and motor neurons in the brain using Ir25a-Gal80, Tsh-Gal80 anism would operate between the initial sensing of sweet food (Clyne and Miesenbo¨ ck, 2008), and Vglut-Gal80 (Bussell et al., and the post-prandial rise in circulating sugar levels. 2014), respectively (Figures S2G and S2H). In these animals, To screen for neurons required for rapid modulation of inges- 57F03> expression was limited to 25 local interneurons in the tion, we genetically inactivated subpopulations of neurons and brain (Figure S2H). To identify the minimum number of neurons monitored ingestion dynamics. Using the Gal4/UAS system responsible for the food ingestion defect, we screened the Jane- (Brand and Perrimon, 1993), we expressed tetanus toxin light lia Farm FlyLight Gal4 Collection (Jenett et al., 2012) for expres- chain, which blocks evoked synaptic transmission by cleaving sion patterns that overlap with 57F03> (Figure 4A) and identified neuronal synaptobrevin (Sweeney et al., 1995). Of 105 viable 83F01>. We used FLP-recombinase-mediated recombination lines tested in the conventional CAFE assay (Ja et al., 2007), to label the 57F03> and 83F01> intersection. FLP controls site- two lines reproducibly showed reduced food intake, and we specific recombination between FRT (FLP recombination target) focused on 57F03-Gal4 (hereinafter, 57F03>)(Figure 3A). sites (Golic and Lindquist, 1989). We confirmed that this method 57F03> tetanus toxin flies ingested less liquid and solid food recapitulated 57F03> expression (Figures S3A and S3B). In the than genetic controls (Figures S2A–S2C) but showed normal brain, the 57F03> and 83F01> intersection selectively labeled proboscis extension to food and normal locomotor/geotaxis 12 interneurons (Figure 4A) with projections limited to the sub- behaviors (Figures S2D–S2F). esophageal zone (Figures 4B and 4C). IN1 processes cross the

(C) Feeding success (chi-square test; ns, not significant; population percentage and 95% confidence intervals). (D) Total ingestion in volume and calories (cyan, mean ± SEM). (E) Total ingestion in nanomoles. (F) Bout volume. p < 0.05, Kruskal-Wallis test, pairwise post hoc comparisons using Tukey and Kramer and (Nemenyi) tests; n = 20. (G) Gray lines show the raw data, and orange lines show the nonlinear regression. (H) Bout rate. p < 0.05, Kruskal-Wallis test, pairwise post hoc comparisons using Tukey and Kramer (Nemenyi) tests; n = 20. (I) Nonlinear regression (orange) ± SEM (gray).

(J) Time to half-total intake (T1/2). p < 0.05, Kruskal-Wallis test, pairwise post hoc comparisons using Tukey and Kramer (Nemenyi) tests; n = 20. Bar plots or box plots labeled with different letters in a given panel are significantly different.

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Figure 3. Genetic Silencing Screen Identifies Neurons that Regulate Ingestion Dynamics (A) Overview of neuronal silencing screen. (B–D) 57F03>CD8-GFP visualized by GFP (green) immunofluorescence in the brain (B), ventral nerve cord (C), and labellum (D). Magenta shows Brp staining of in (B) and (C) and anti-Ir25a antibody in (D) (scale bars, 20 mm). (E–J) Expresso measurement of 24-hr-fasted male flies of indicated genotypes consuming 1 M sucrose (n = 20). (E) Meal-bout raster plots. (F) Total ingestion of all flies per 50-s bin over a 33-min experiment. (G) Feeding success (chi-square test, pairwise post hoc comparisons with Fisher’s exact test with Bonferroni correction; ns, not significant; population percentage and 95% confidence interval). (H) Total sucrose ingestion volume, (I) bout volume, and (J) bout rate. In (H)–(J), p < 0.05, Kruskal-Wallis test, pairwise post hoc comparisons using Tukey and Kramer (Nemenyi) test; n = 20. Error bars indicate mean ± SEM. Bar plots or box plots labeled with different letters in a given panel are significantly different. See also Figure S2.

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Figure 4. IN1 Cholinergic Local Taste Interneurons Regulate Sucrose Ingestion (A) Schematic of the 57F03> and 83F01> (IN1) intersection (left). Quantification of GFP-positive cells in the IN1 intersection (n = 6 brains) (right). Error bars indicate mean ± SEM. (B) IN1>CD8-GFP (GFP, green) and Neuropil (Brp, magenta) (scale bar, 20 mm). (C) Gray scale image of the boxed region in (B) (scale bar, 20 mm). (D) A single IN1 neuron labeled by GFP (scale bar, 20 mm). (E–J) Expresso measurement of 24-hr-fasted male flies of the indicated genotypes consuming 1 M sucrose (n = 20). (E) Meal-bout raster plots. (F) Total ingestion of all flies per 50-s bin over 33-min experiment. (G) Feeding success (chi-square test, pairwise post hoc comparisons with Fisher’s exact test with Bonferroni correction; ns, not significant; population percentage and 95% confidence interval are shown). (H) Total ingestion, (I) bout volume, and (J) bout rate. p < 0.05, Kruskal-Wallis test, pairwise post hoc comparisons using Tukey and Kramer (Nemenyi) tests; n = 20. Bar plots or box plots labeled with different letters in a given panel are significantly different. See also Figure S3.

Cell 165, 715–729, April 21, 2016 721 A B Figure 5. IN1 Neurons Receive Presynaptic Input from Sugar-Sensitive Neurons in the Pharynx (A and B) Fly brain (gray) and proboscis (gray) schematics. Boxes indicate the taste center (sub- esophageal zone; cyan), the labral organ, and the labellum. (C–H) Taste neuron afferents (magenta) and IN1 interneurons (green) in the anterior (left) and pos- terior (right) subesophageal zone (scale bars, 20 mm). The Gr-Gal4 lines are Gr66a (C), Gr5a (D), Gr64f (E), Gr61a (F), Gr43a (G), and Gr64a (H). C I (I–N) Taste neurons labeled by Gr-Gal4 lines in the labral sense organ (left, magenta) and in the labellum (right, magenta) (scale bars, 20 mm). The Gr-Gal4 lines are Gr66a (I), Gr5a (J), Gr64f (K), Gr61a (L), Gr43a (M), and Gr64a (N). D J (O) Schematic of synaptic GRASP between IN1 interneurons (gray) and taste neurons (magenta). (P) Normalized GRASP signal (scale bars, 1 mm) (*p < 0.05, unpaired Student’s t test; ns, not sig- nificant; error bars indicate mean ± SEM, n = 3). E K See also Figure S4.

L F controls (Figures 4E–4G), but total inges- tion, meal-bout volume, and ingestion rate were dramatically reduced (Figures 4H–4J). G M To characterize the polarity and neuro- chemistry of IN1 neurons, we labeled them with markers enriched in (GFP fused to synaptotagmin) or H N (GFP fused to Dscam) (Wang et al., 2004; Zhang et al., 2002). Synaptotagmin-GFP and Dscam-GFP labeled overlapping areas within the subesophageal zone (Figures S4A and S4B). IN1 neurons did not express dopaminergic, serotonergic, O P glutamatergic, or GABAergic markers (data not shown). However, IN1> expres- sion was blocked by Gal80 expression in cholinergic cells, suggesting that these neurons are cholinergic (Figures S4C and S4D). Acetylcholine is the major excitatory neurotransmitter in the Drosophila brain.

IN1 Interneurons Receive Presynaptic Input from Sugar- Sensitive Neurons in the Pharynx Our behavioral and anatomical experi- ments suggest that IN1 neurons consti- tute a previously uncharacterized class midline and arborize in both ipsilateral and contralateral sides of of cholinergic local interneurons in the fly taste center required the subesophageal zone (Figure 4D). The IN1 intersection strain for sustained sucrose ingestion. We characterized sensory input also labeled a pair of ascending neurons in the ventral nerve cord to IN1 from the taste periphery (Figures 5A and 5B) with Gr-Gal4 (Figure S3C), but the functional relevance of this structure strains to label neurons expressing sweet (Chyb et al., 2003; Da- for ingestion was previously excluded (Figures S2G and S2H). hanukar et al., 2007; Miyamoto et al., 2012) or bitter (Weiss et al., The percentage of 24-hr-fasted IN1> tetanus toxin flies that 2011) taste receptors. The axons of bitter and sweet taste neu- consumed 1 M sucrose in Expresso did not differ from that of rons with cell bodies in the labellum did not overlap with IN1

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E 1 M sucrose 100mM sucrose F G aa,bb aab 3.0 3.0 600 Fed 24hr fasted 24hr fasted 0 0 1.75 1.5 F/F ∆F/F (A.U.C) Persistent activity 0.5 0 0 Ingestion Fed Fasted Fasted Fed Fasted Fasted

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(legend on next page) Cell 165, 715–729, April 21, 2016 723 neurons (Figures 5C–5F and 5I–5L, right panels). In contrast, GCaMP6s fluorescence in IN1 neuron processes in fed and fasted afferents from sugar-sensitive taste neurons that line the labral flies that were offered a single 20-nl drop of 1 M sucrose. 1 M su- organ of the pharynx overlapped with IN1 interneuron arbors crose ingestion elicited significantly higher stimulus-evoked ac- (Figures 5E–5H and 5K–5N, left panels). tivity in IN1 neurons in fasted compared to fed flies (Figures 6E– To determine whether IN1 neurons are synaptically connected 6G). In fasted flies, 1 M sucrose-evoked activity of IN1 neurons to afferent sensory axons from the pharynx, we used a modifi- lasted for at least 7 min, decaying to 57% of the stimulus-evoked cation of the GFP Reconstitution Across Synaptic Partners maximum F/F0 (SEM: ±5.7). However in fed flies, this activity was (GRASP) method (Feinberg et al., 2008) to label synaptic sites not sustained. When fasted flies ingested 100 mM sucrose, the selectively (Chen et al., 2014)(Figure 5O). There was a significant evoked GCaMP6s response was similar in amplitude to 1 M GRASP signal between IN1 interneurons and sugar-sensitive sucrose ingestion, but the response was transient, with fluores- pharyngeal taste neurons labeled by Gr43a and Gr64a (Fig- cence quickly returning to baseline (Figure 6E). Thus, both hunger ure 5P). We detected no GRASP signal between IN1 interneu- state and sucrose concentration modulate IN1 activity. rons and Gr5a sweet taste neurons on the labellum (Figure 5P). If IN1 neurons regulate the intensity of sucrose ingestion in hun- We conclude that IN1 interneurons receive specific input from gry flies, IN1 activity should decrease steadily, as cumulative su- sweet taste neurons in the pharynx. crose ingestion satiates the fly. To test this hypothesis, we offered 24-hr-fasted flies 20-nl drops of 1 M sucrose repeatedly. In these IN1 Activation by Sucrose Ingestion and Its Modulation real-time sucrose ingestion/imaging experiments, the first inges- by Hunger State tion event induced a large and persistent increase in GCaMP6s Next, we asked whether IN1 neurons respond to sucrose inges- activity in IN1 neurons (Figure 6H). Subsequent ingestion events tion and whether this activation is altered by satiation, as pre- evoked neuronal activity on top of this elevated baseline. As the dicted by the behavioral and anatomical results presented earlier. experiment progressed, the fly became less likely to consume We used in vivo two-photon imaging of head-fixed flies express- the sucrose stimulus (Figure 6H). Initial full-volume ingestion ing the genetically encoded calcium sensor GCaMP6s in IN1 neu- events transitioned into half-volume events and concluded with rons (Figures S5A and S5B). Rapid volumetric imaging of the a series of minute-volume or failed ingestions. In parallel, evoked GCaMP6s fluorescence in the IN1 neuronal arbor was monitored IN1 responses decreased across these trials (Figures 6H and 6I). while 24-hr-fasted flies were offered a drop of 1 M sucrose or wa- ter (Figures 6A and 6D; Movie S2). IN1 neurons did not respond Activation of IN1 Suffices to Trigger Sucrose Ingestion when the proboscis touched the sucrose drop or when flies Fed flies ingest little or no food. If the activity of IN1 neurons reg- ingested water but were strongly activated when 1 M sucrose ulates sucrose ingestion in hungry flies, artificial activation of was ingested (Figures 6A–6D). Because we did not image these neurons should suffice to trigger food ingestion in satiated GCaMP6s activity in individual IN1 cell bodies, we cannot exclude animals. We expressed the channelrhodopsin variant ReaChR the possibility that only some of these neurons responded to (Inagaki et al., 2014a) in sweet taste neurons or IN1 neurons (Fig- sucrose ingestion. In the example shown in Figures 6A and 6B, ures S6A–S6C) and monitored behavior in response to photoac- the fasted fly consumed the entire 20-nl drop in one continuous tivation. Photoactivation of sweet taste neurons (Gr5a>ReaChr) bout. As the fly drank, GCaMP6s activity rose rapidly and re- reliably triggered robust proboscis extension that was tempo- mained elevated throughout the ingestion bout, which lasted, on rally controlled by the light pulse (Figures 7A and 7B; Movie average, 10.4 s per fly. Remarkably, this activity persisted after S3). In contrast, IN1>ReaChR flies did not show proboscis the fly stopped ingesting 1 M sucrose (Figures 6A and 6B). extension upon photoactivation when food was not present (Fig- To explore the effects of hunger state and sucrose concen- ures 7A and 7B). However, when flies were placed in close prox- tration on IN1 responses and persistent activity, we measured imity to a drop of 1 M sucrose (Figure S6D), photoactivation

Figure 6. IN1 Activity Integrates Hunger State and Sucrose Concentration (A) Representative IN1>GCaMP6s responses recorded in the same 24-hr-fasted female before, during, and after 1 M sucrose ingestion. Still images captured by the video camera (a–d; see B for corresponding raw traces), with the eye pseudocolored in red and 1 M sucrose drop in blue (top). Heatmap of IN1 neuron activity in response to indicated stimuli (bottom). (B) Trace of IN1 fluorescence in a.u., with letters a–d indicating the corresponding still image and activity heatmaps in (A).

(C) Fluorescence traces are normalized using F0. The gray lines show data from individual flies; bold green and blue lines show average traces for the indicated stimuli. (D) Peak of stimulus-evoked IN1 neuron activity (p < 0.05, one-way ANOVA with pairwise post hoc Bonferroni test; error bars indicate mean ± SEM; n = 4–6). (E) Normalized IN1 neuron activity in fed or 24-hr-fasted flies to 1 M or 100 mM sucrose. Mean traces for indicated stimuli and conditions (colored lines) ± SEM (gray). Summed histogram of ingestion duration (bottom). (F) Peak of stimulus-evoked IN1 neuron responses to indicated stimuli and conditions (p < 0.05, unpaired Student’s t test with Bonferroni correction; error bars indicate mean ± SEM; n = 5–6). (G) Area under the curve (A.U.C.) measurement of the GCaMP6s signal showing the persistent activity of IN1 neurons to indicated stimuli and conditions (p < 0.05, Student’s t test with Bonferroni correction; error bars indicate mean ± SEM, n = 5–6). (H) Normalized IN1 neuron responses to repeated 1 M sucrose stimulation. GCaMP6s fluorescence traces and episodes of 1 M sucrose ingestion are plotted per trial. (I) Normalized IN1 responses binned by the percent number of trials, but excluding the first ingestion (**p < 0.01, F test for linear regression; error bars indicate mean ± SEM, n = 3). Bar plots or box plots labeled with different letters in a given panel are significantly different. See also Figures S5A and S5B and Movie S2.

724 Cell 165, 715–729, April 21, 2016 A B Figure 7. Photoactivation of IN1 Neurons Stimulates Sucrose Ingestion in Fed Flies (A) Proboscis extension response of indicated genotypes in response to photoactivation in the absence of food (chi-square test; ns, not significant; population percentage and 95% confidence in- tervals, n = 15). (B) Gr5a-ReaChR and IN1-ReaChR male flies during photoactivation, no food present. (C and D) Fed (0 hr fasted) control (C) and IN1-ReaChR (D) male flies before (left) and during C D (right) photoactivation. (E and F) Raster plots of 1 M sucrose ingestion by control (E) and IN1-ReaChR (F) male flies before (left) and during (right) photoactivation (n = 15 per geno- type). The horizontal black bars show the time and duration of individual meal-bouts ingested by single flies. (G and H) Time-series data of 1 M sucrose ingestion E F by control (G) and IN1-ReaChR (H) male flies before (left) and during (right) photoactivation (n = 15 for each genotype). Population averages are smooth- ened by the LOWESS method and plotted against time. The green traces represent the light pulses used for photoactivation (bottom). (I) Duration of 1 M sucrose ingestion of indicated genotypes and conditions (***p < 0.001, Wilcoxon signed-rank test; ns, not significant; error bars indi- GHcate mean ± SEM, n = 15). (J) Estimated volume measurements of 1 M sucrose ingestion of indicated genotypes and conditions (***p < 0.001, Wilcoxon signed-rank test; ns, not significant; error bars indicate mean ± SEM, n = 15). Bar plots or box plots labeled with different letters in a given panel are significantly different. See also Figure S6 and Movie S3.

IJ

1976; Morton et al., 2006). We have discovered 12 cholinergic interneurons in the taste processing center of the fly brain that tune the dynamics of sucrose inges- tion. Our data support a model in which sweet taste neurons in the pharynx proj- ect to the subesophageal zone, where they contact IN1 interneurons. When a increased the probability of food ingestion in IN1>ReaChR flies, hungry animal first drinks sucrose, pharyngeal sweet taste neu- but not in controls (Figures 7C–7H). Out of 15 IN1>ReaChr flies rons are stimulated and activate IN1 neurons. In hungry flies, this tested, 11 exhibited intermittent ingestion throughout the light leads to persistent IN1 activity, which, in turn, sustains ingestion. stimulation period (Figure 7F). We estimated the ingested vol- IN1 activity in fed flies or those offered a lower sucrose concen- ume as the fly fed (Figures S6E and S6F). Photoactivation tration is attenuated, and these animals do not show sustained increased the volume and duration of 1 M sucrose ingestion in ingestion. As the fly becomes satiated, IN1 neurons become IN1>ReaChR flies but not in controls (Figures 7I and 7J). Light- insensitive to sucrose, which decreases the drive to ingest, stimulated ingestion events of IN1>ReaChR flies were not time and results in shorter ingestion episodes. We propose that the locked to photoactivation. Instead, IN1 activation increased the IN1 circuit provides a fast feedback mechanism to regulate probability that flies ingest 1 M sucrose. sucrose ingestion by integrating taste and hunger signals.

DISCUSSION Functional Characterization of a Drosophila Ingestion Circuit The neural circuits that integrate taste, hunger, and metabolism Although previous work investigating the dynamics of fly feeding to control food ingestion remain poorly understood (Dethier, used proboscis extension as a proxy for food intake (Inagaki

Cell 165, 715–729, April 21, 2016 725 et al., 2012, 2014b; Kain and Dahanukar, 2015; Marella et al., ansen, 1967; Ishimaru et al., 2005; Travers and Nicklas, 1990). 2012), this pre-ingestive response is not a reliable predictor of In zebrafish, the sweet taste receptors T1R2/T1R3 are ex- ingestion (Deshpande et al., 2014). In classic work on the blowfly, pressed in the pharynx, as well as in the and gill raker (Ishi- Vincent Dethier suggested that food consumption is controlled by maru et al., 2005). Expression of T1R2/T1R3 in the pharynx factors that stimulate ingestion rather than those that act on pe- has not been investigated in mammals, but it is known that the ripheral taste or post-ingestive nutrient sensing (Deth- cranial nerve fibers innervating pharyngeal taste cells respond ier et al., 1956). Since IN1 neurons are activated when sucrose is to sugar (Frank, 1991; Hanamori et al., 1988). These neurons ingested, and their activity is modulated by both hunger state project to the nucleus of (NST) in the hindbrain (Car- and taste stimuli, they are uniquely situated to modulate ingestion. leton et al., 2010; Frank, 1991; Hanamori et al., 1988). The NST In support of this, our functional imaging experiments revealed also receives peripheral satiety signals transmitted via vagal that a single bout of sucrose ingestion by hungry flies induced acti- nerve fibers, indicating that this brain region is involved in vation of IN1 neurons that persisted for at least 7 min. Long-lasting many regulatory processes that regulate food intake, including changes in the activity of hypothalamic AgRP and POMC neurons the decision to continue ingestion (Carleton et al., 2010; Morton have been previously linked to the regulation of state-dependent et al., 2006). Based on the similarities of insect and vertebrate feeding decisions in mice (Betley et al., 2015; Chen et al., 2015). taste-processing systems, we speculate that interneuron popu- Likewise, persistent neural activity in IN1 neurons may prepare lations in the NST with the physiological properties of IN1 neu- the nervous system in hungry flies for further food ingestion. rons may exist. Taste perception by the pharynx would permit What are the origins of the persistent activity seen in IN1 rapid assessment of food intake volume and quality and provide neurons? Intrinsic or extrinsic factors may contribute. The area real-time feedback to the central brain to regulate ingestion in where IN1 processes arborize receives input from dopaminergic, vertebrates as well as in insects. serotonergic, and peptidergic afferents (data not shown). These neuromodulatory circuits may be sensitive to satiety state (Albin EXPERIMENTAL PROCEDURES et al., 2015; Marella et al., 2012) and help to prolong IN1 activity elicited by sucrose ingestion in fasted animals. It is plausible Detailed methods associated with all procedures discussed in the following that, once the fly reaches satiety, neuromodulatory activity is text are available in the Supplemental Experimental Procedures. tempered, and IN1 neurons return to their basal state. Alterna- tively, IN1 neurons may have unique intrinsic biophysical fea- Fly Strains Simplified genotypes of fly strains are provided in the main text and figures for tures that sustain their activity after sucrose stimulation. A clarity. complete characterization of IN1 membrane excitability and circuitry is necessary to investigate these ideas. Behavior All behavioral assays were carried out in a 23–25C incubator, under a Connectivity in Feeding Circuits 12-hr:12-hr light:dark cycle (lights on at 9 a.m.) and 50%–60% relative humid- Recent studies have identified a number of functionally distinct ity, unless stated otherwise. All assays were performed at zeitgeber time 5–10 populations of neurons in the fly taste circuit that regulate different with 3- to 10-day-old males, unless stated otherwise. aspects of food intake behavior. Neuropeptide F and dopamine CAFE Food Intake Screen This assay was modified from earlier studies (Ja et al., 2007). To measure post- signaling enhance the responsiveness of labellar taste sensory fasting food intake, 24-hr-fasted flies were given access to liquid food for 3 hr. neurons in hungry flies and increase the probability of initiating For each genotype in the screen (Figure 3A), a group of ten flies was tested. food intake (Inagaki et al., 2012, 2014b; Marella et al., 2012). How- Locomotion Assay ever, when labellar sweet taste perception is perturbed, ingestion Fly locomotion was tracked in a previously described custom-made 70-mm is not affected (LeDue et al., 2015). Thus, labellar taste neuron cir- circular arena (Simon and Dickinson, 2010) modified by Bussell et al. (2014), cuitry likely regulates initial food evaluation but not the later deci- with 10–15 flies per genotype tested. Negative Geotaxis sion to ingest food. In contrast, IN1 activity is required specifically This assay was modified from a previously described protocol (Stafford et al., to sustain sucrose ingestion. This makes IN1 neurons functionally 2012). and anatomically distinct from all previously described neural Proboscis Extension pathways that mediate taste responsiveness and feeding initiation. Procedures were modified from a previously described protocol (Shiraiwa and Previous work has identified interneurons that regulate the feeding Carlson, 2007). motor program (Flood et al., 2013), GABAergic neurons that sup- Solid Food Intake press nonselective ingestion (Pool et al., 2014), and motor neurons Ten flies were wet-fasted and then given access to solid food in a polystyrene vial (Fisher #AS-519) for 15 min. Flies were scored as fully fed only if their that regulate fluid ingestion (Manzo et al., 2012). How these neu- abdomen was completely filled with green food. rons connect taste sensory input to the motor output of ingestion Expresso Feeding System as well as interpret top-down information about hunger state is Assay hardware was designed and constructed in consultation with William not known. We propose that IN1 neurons participate in this Dickson at IO Rodeo. The Expresso data analysis software was written in circuit as a key node that governs rapid food intake decisions. MATLAB 2013a (MathWorks). Photoactivation IN1>ReaChR-expressing flies were raised on standard fly food at 25C A Conserved Mechanism for Ingestion Sensing via and 50%–60% relative humidity in an incubator. 3- to 5-day-old male Pharyngeal Taste? flies were collected and housed in groups of 10–20 with food containing In vertebrates, taste cells are present not only in the tongue but 400 mM all-trans-retinal (Sigma-Aldrich #R2500) mixed into standard fly also in other organs, including the pharynx (Henkin and Christi- food. The flies were kept at 25C and 50%–60% relative humidity in an

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