Animals must set behavioural priority in a context-dependent manner and switch from one behaviour to another at the appropriate moment1,2,3. Here we probe the molecular and neuronal mechanisms that orchestrate the transition from feeding to courtship in Drosophila melanogaster. We find that feeding is prioritized over courtship in starved males, and the consumption of protein-rich food rapidly reverses this order within a few minutes. At the molecular level, a gut-derived, nutrient-specific neuropeptide hormone—Diuretic hormone 31 (Dh31)—propels a switch from feeding to courtship. We further address the underlying kinetics with calcium imaging experiments. Amino acids from food acutely activate Dh31+ enteroendocrine cells in the gut, increasing Dh31 levels in the circulation. In addition, three-photon functional imaging of intact flies shows that optogenetic stimulation of Dh31+ enteroendocrine cells rapidly excites a subset of brain neurons that express Dh31 receptor (Dh31R). Gut-derived Dh31 excites the brain neurons through the circulatory system within a few minutes, in line with the speed of the feeding–courtship behavioural switch. At the circuit level, there are two distinct populations of Dh31R+ neurons in the brain, with one population inhibiting feeding through allatostatin-C and the other promoting courtship through corazonin. Together, our findings illustrate a mechanism by which the consumption of protein-rich food triggers the release of a gut hormone, which in turn prioritizes courtship over feeding through two parallel pathways.
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Tinbergen, N. The Study of Instinct (Clarendon Press, 1951).
McFarland, D. J. Decision making in animals. Nature 269, 15–21 (1977).
Stearns, S. C. The Evolution of Life Histories (Oxford Univ. Press, 1992).
Sutton, A. K. & Krashes, M. J. Integrating hunger with rival motivations. Trends Endocrinol. Metab. 31, 495–507 (2020).
Marella, S., Mann, K. & Scott, K. Dopaminergic modulation of sucrose acceptance behavior in Drosophila. Neuron 73, 941–950 (2012).
Morton, G. J., Meek, T. H. & Schwartz, M. W. Neurobiology of food intake in health and disease. Nat. Rev. Neurosci. 15, 367–378 (2014).
Inagaki, H. K. et al. Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing. Cell 148, 583–595 (2012).
Hadjieconomou, D. et al. Enteric neurons increase maternal food intake during reproduction. Nature 587, 455–459 (2020).
Karigo, T. et al. Distinct hypothalamic control of same- and opposite-sex mounting behaviour in mice. Nature 589, 258–263 (2021).
Bayless, D. W. et al. Limbic neurons shape sex recognition and social behavior in sexually naive males. Cell 176, 1190–1205.e20 (2019).
Yang, C. F. et al. Sexually dimorphic neurons in the ventromedial hypothalamus govern mating in both sexes and aggression in males. Cell 153, 896–909 (2013).
Dickson, B. J. Wired for sex: the neurobiology of Drosophila mating decisions. Science 322, 904–909 (2008).
Yamamoto, D., Sato, K. & Koganezawa, M. Neuroethology of male courtship in Drosophila: from the gene to behavior. J. Comp. Physiol. A 200, 251–264 (2014).
Zhang, S. X., Rogulja, D. & Crickmore, M. A. Dopaminergic circuitry underlying mating drive. Neuron 91, 168–181 (2016).
Piper, M. D. W. et al. A holidic medium for Drosophila melanogaster. Nat. Methods 11, 100–105 (2014).
Cheriyamkunnel, S. J. et al. A neuronal mechanism controlling the choice between feeding and sexual behaviors in Drosophila. Curr. Biol. 31, 4231–4245.e4 (2021).
Lin, H.-H. et al. Hormonal modulation of pheromone detection enhances male courtship success. Neuron 90, 1272–1285 (2016).
Schneider, J. E., Wise, J. D., Benton, N. A., Brozek, J. M. & Keen-Rhinehart, E. When do we eat? Ingestive behavior, survival, and reproductive success. Horm. Behav. 64, 702–728 (2013).
Guo, X. et al. The cellular diversity and transcription factor code of Drosophila enteroendocrine cells. Cell Rep. 29, 4172–4185.e5 (2019).
Hung, R.-J. et al. A cell atlas of the adult Drosophila midgut. Proc. Natl Acad. Sci. USA 117, 1514–1523 (2020).
Veenstra, J. A. & Ida, T. More Drosophila enteroendocrine peptides: Orcokinin B and the CCHamides 1 and 2. Cell Tissue Res. 357, 607–621 (2014).
Park, J.-H. et al. A subset of enteroendocrine cells is activated by amino acids in the Drosophila midgut. FEBS Lett. 590, 493–500 (2016).
Bellen, H. J. et al. The BDGP Gene Disruption Project. Genetics 167, 761–781 (2004).
Asahina, K. et al. Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila. Cell 156, 221–235 (2014).
Hergarden, A. C., Tayler, T. D. & Anderson, D. J. Allatostatin-A neurons inhibit feeding behavior in adult Drosophila. Proc. Natl Acad. Sci. USA 109, 3967–3972 (2012).
Clyne, J. D. & Miesenböck, G. Sex-specific control and tuning of the pattern generator for courtship song in Drosophila. Cell 133, 354–363 (2008).
Marella, S. et al. Imaging taste responses in the fly brain reveals a functional map of taste category and behavior. Neuron 49, 285–295 (2006).
Jordt, S.-E. & Julius, D. Molecular basis for species-specific sensitivity to ‘hot’ chili peppers. Cell 108, 421–430 (2002).
Klapoetke, N. C. et al. Independent optical excitation of distinct neural populations. Nat. Methods 11, 338–346 (2014).
Johnson, E. C. et al. A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling. J. Exp. Biol. 208, 1239–1246 (2005).
Dana, H. et al. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nat. Methods 16, 649–657 (2019).
Tao, X. et al. Transcutical imaging with cellular and subcellular resolution. Biomed. Opt. Express 8, 1277–1289 (2017).
Badre, N. H., Martin, M. E. & Cooper, R. L. The physiological and behavioral effects of carbon dioxide on Drosophila melanogaster larvae. Comp. Biochem. Physiol. A 140, 363–376 (2005).
Masuyama, K., Zhang, Y., Rao, Y. & Wang, J. W. Mapping neural circuits with activity-dependent nuclear import of a transcription factor. J. Neurogenet. 26, 89–102 (2012).
Wu, Q. et al. Excreta quantification (EX-Q) for longitudinal measurements of food intake in Drosophila. iScience 23, 100776 (2020).
Al-Anzi, B. et al. The leucokinin pathway and its neurons regulate meal size in Drosophila. Curr. Biol. 20, 969–978 (2010).
Clark, L., Zhang, J. R., Tobe, S. & Lange, A. B. Proctolin: a possible releasing factor in the corpus cardiacum/corpus allatum of the locust. Peptides 27, 559–566 (2006).
Down, R. E., Matthews, H. J. & Audsley, N. Effects of Manduca sexta allatostatin and an analog on the pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae) and degradation by enzymes from the aphid gut. Peptides 31, 489–497 (2010).
Gáliková, M., Dircksen, H. & Nässel, D. R. The thirsty fly: Ion transport peptide (ITP) is a novel endocrine regulator of water homeostasis in Drosophila. PLoS Genet. 14, e1007618 (2018).
Min, S. et al. Identification of a peptidergic pathway critical to satiety responses in Drosophila. Curr. Biol. 26, 814–820 (2016).
Scopelliti, A. et al. A neuronal relay mediates a nutrient responsive gut/fat body axis regulating energy homeostasis in adult Drosophila. Cell Metab. 29, 269–284.e10 (2019).
Söderberg, J. A. E., Carlsson, M. A. & Nässel, D. R. Insulin-producing cells in the Drosophila brain also express satiety-inducing cholecystokinin-like peptide, drosulfakinin. Front. Endocrinol. 3, 109 (2012).
Wu, Q. et al. Developmental control of foraging and social behavior by the Drosophila neuropeptide Y-like system. Neuron 39, 147–161 (2003).
Pfeiffer, B. D. et al. Refinement of tools for targeted gene expression in Drosophila. Genetics 186, 735–755 (2010).
Ng, R. et al. Amplification of Drosophila olfactory responses by a DEG/ENaC channel. Neuron 104, 947–959.e5 (2019).
Maslow, A. H. A theory of human motivation. Psychol. Rev. 50, 370–396 (1943).
Alhadeff, A. L. et al. A neural circuit for the suppression of pain by a competing need state. Cell 173, 140–152.e15 (2018).
Saper, C. B., Fuller, P. M., Pedersen, N. P., Lu, J. & Scammell, T. E. Sleep state switching. Neuron 68, 1023–1042 (2010).
Jourjine, N., Mullaney, B. C., Mann, K. & Scott, K. Coupled sensing of hunger and thirst signals balances sugar and water consumption. Cell 166, 855–866 (2016).
Kondo, S. & Ueda, R. Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics 195, 715–721 (2013).
Sakai, T., Isono, K., Tomaru, M. & Oguma, Y. Light-affected male following behavior is involved in light-dependent mating in Drosophila melanogaster. Genes Genet. Syst. 72, 275–281 (1997).
Itskov, P. M. et al. Automated monitoring and quantitative analysis of feeding behaviour in Drosophila. Nat. Commun. 5, 4560 (2014).
Veenstra, J. A., Agricola, H.-J. & Sellami, A. Regulatory peptides in fruit fly midgut. Cell Tissue Res. 334, 499–516 (2008).
Peabody, N. C. et al. Bursicon functions within the Drosophila CNS to modulate wing expansion behavior, hormone secretion, and cell death. J. Neurosci. 28, 14379–14391 (2008).
Goda, T. et al. Calcitonin receptors are ancient modulators for rhythms of preferential temperature in insects and body temperature in mammals. Genes Dev. 32, 140–155 (2018).
Kunst, M. et al. Calcitonin gene-related peptide neurons mediate sleep-specific circadian output in Drosophila. Curr. Biol. 24, 2652–2664 (2014).
Wang, J. W., Wong, A. M., Flores, J., Vosshall, L. B. & Axel, R. Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain. Cell 112, 271–282 (2003).
Pnevmatikakis, E. A. & Giovannucci, A. NoRMCorre: An online algorithm for piecewise rigid motion correction of calcium imaging data. J. Neurosci. Methods 291, 83–94 (2017).
Dorostkar, M. M., Dreosti, E., Odermatt, B. & Lagnado, L. Computational processing of optical measurements of neuronal and synaptic activity in networks. J. Neurosci. Methods 188, 141–150 (2010).
Lindsay, S. A., Lin, S. J. H. & Wasserman, S. A. Short-form bomanins mediate humoral immunity in Drosophila. J. Innate Immun. 10, 306–314 (2018).
Lee, J., Iyengar, A. & Wu, C.-F. Distinctions among electroconvulsion- and proconvulsant-induced seizure discharges and native motor patterns during flight and grooming: quantitative spike pattern analysis in Drosophila flight muscles. J. Neurogenet. 33, 125–142 (2019).
Klassen, M. P. et al. Age-dependent diastolic heart failure in an in vivo Drosophila model. eLife 6, e20851 (2017).
Dus, M. et al. Nutrient sensor in the brain directs the action of the brain–gut axis in Drosophila. Neuron 87, 139–151 (2015).
Branon, K., Robie, A. A., Bender, J., Perona, P. & Dickinson, M. H. High-throughput ethomics in large groups of Drosophila. Nat. Methods 6, 451–457 (2009).
We thank D. Kleinfeld, C.-Y. Su and K. Asahina for comments on the manuscript; Y. Jin for sharing the spectrophotometer; D. Kleinfeld and C. Xu for continuous advice on three-photon microscopy; the following individuals for reagents: J. Veenstra (Dh31, AstB and AstC antiserum), B. White (Bursicon antiserum), F. Hamada (Dh31R antiserum) and R. Bodmer (R94C02::tdTomato transgenic line); and members of the Wang laboratory and the Su laboratory for stimulating discussions. Confocal imaging was supported by NINDS P30 NS047101 (UC San Diego Neuroscience Microscopy Imaging Core). I.H. and M.R. acknowledge funding from the National Science Foundation (1429810). This work was supported by NIH grants to J.W.W. (R01DK127516, R01DK092640 and R01DC009597) and to H.-H.L. (K99DC016338).
The authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Amino acid refeeding promotes the transition from feeding to courtship in starved males.
(a–b) Starvation reduces courtship activity. Arena size, Ø35mm. Raster plots show second-by-second behaviors of individual males (a). Left, males starved for 24 h. Right, males ad libitum fed with standard fly food containing protein and carbohydrates. Fraction of time each male spent on courtship (b). Two-tailed t-test, n = 10 males per condition. (c–d) Consumption of amino acids promotes male courtship. Arena size, Ø20mm. The raster plots show second-by-second behaviors (top panel in c). Four conditions (from left to right): 1) males starved for 24 h before test, in an arena without food; 2) males ad libitum fed with standard fly food containing protein and carbohydrates before test, in an arena without food; 3) males starved for 24 h before test, in an arena with sucrose-containing food; 4) males starved for 24 h before test, in an arena with amino acids-containing food. Each arena also contained one virgin female. Average fraction of time on feeding, courtship and grooming is plotted against time in solid lines in 2-min time bin with SEM as shaded areas (bottom panel in c). (d) Fraction of time (during the period of 10–30 min) each male spent on feeding, courtship and grooming. Time after the start of copulation was excluded from calculation. Two-tailed t-test (left panel in d). Significant differences (p < 0.05) are indicated by different letters, One-way ANOVA followed by Tukey’s test (d). n = 10 flies for each condition. Detailed statistical analysis is available in Supplementary Tables 7. All box and whisker plots show min, max, lower quartile, upper quartile and median.
(a–c) Male flies exhibit similar locomotion activity after consuming food containing isocaloric sucrose or amino acids. After deprived of food for 24 h, individual flies were given access to food for 10 min. Male flies were then placed in a circular arena (60 mm in diameter, 3.5 in mm depth) for video recordings. Food: sucrose (50 mM) or amino acids (200 mM). (a) Walking trajectories of individual flies during the 5-min observation period. (b) Average walking velocity of individual flies. n = 10 flies for each food. Two-tailed t-test. (c) Average path length traveled by flies. n = 10 flies for each condition. (d–f) Gut Dh31 knockdown does not affect locomotion activity. Ctrl1: Dh31-GAL4, Tsh-GAL80. Ctrl2: UAS-Dh31-RNAi. Gut Dh31 KD flies contain all three transgenes. (d) Walking trajectories of individual flies. (e) Average walking velocity of individual flies during the 5-min observation period. n = 10 flies for each genotype. One-way ANOVA test. (f) Average path length traveled by flies. n = 10 flies for each genotype.
(a) A T2A-GAL4.2 cassette was inserted at the C-terminus of Dh31 by Cas9-mediated knock-in strategy. 3XP3-RFP was used as a positive selection marker. (b) An nls-LexA::p65 cassette was inserted after the start codon of Dh31R by Cas9-mediated knock-in strategy. (c) An nls-LexA::p65 cassette was inserted after the start codon of Crz by Cas9-mediated knock-in strategy.
Extended Data Fig. 4 Dh31 expression outside the brain is required for the effect of ingested amino acids on courtship.
(a). Validation of the Dh31T2A-GAL4 driver line by a Dh31 antiserum. Representative confocal images show the expression pattern of Dh31T2A-GAL4 (Blue) and Dh31 (magenta) in the brain, ventral nerve cord (VNC) and midgut of male flies carrying Dh31T2A-GAL4 and UAS-Redstinger. Schematic indicates the distribution of Dh31 cells in the brain, VNC, and GI tract, drawn from the confocal stacks of the sample shown in the left panel. Magenta dots show cell positive for Dh31 antibody and Redstinger; black dots show cells positive for Dh31 antibody and negative for Redstinger. The average number (± SEM) of cells in each region is from 5 different male flies. Scale bar, 50 μm. (b–d) The courtship effect of region-specific Dh31 knockdown. Dh31 knockdown in all Dh31T2A-GAL4 cells (b); Dh31 knockdown in brain Dh31T2A-GAL4 cells (c); Dh31 knockdown in Dh31T2A-GAL4 cells outside the brain (d). Representative images from 3 samples. For each condition, the total number of matches (n) from 5 independent experiments is indicated in the figure. p-value was determined by Chi-square test to indicate whether males of a given genetic manipulation respond to amino acids. Blue bar indicates the average copulation percentage, and the dashed line indicates chance level. AAs, amino acids (200 mM). S, sucrose (50 mM).
Extended Data Fig. 5 Dh31 expression in enteroendocrine cells is required for the effect of ingested amino acids.
(a) Expression of Redstinger (blue) and Dh31 (anti-Dh31, magenta) in the brain, VNC and midgut of a male containing Dh31-GAL4 and UAS-Redstinger. Scale bar, 50 μm. Schematic: cell distribution in the brain, VNC and GI tract, drawn from the confocal stacks of the sample shown in the left panel. Magenta dots: Dh31 antibody+ and GAL4+ cells; black dots: Dh31 antibody+ and GAL4− cells. The average number (± SEM) of cells in each region is from 5 different male flies. (b) Validation of Dh31 knockdown. Knockdown of Dh31 in the gut is achieved by using Dh31-GAL4 and Tsh-GAL80 to drive UAS-Dh31-RNAi. Immunostaining with an antiserum to Dh31 (magenta) shows that knockdown is effective (3 different experiments). Scale bar, 50 μm. (c). The courtship effect of gut-specific Dh31 knockdown (left) or chemo-activation of gut Dh31+ cells (right). Chemo-activation is achieved by using Dh31-GAL4 and Tsh-GAL80 to drive UAS-VR1. Each data point represents one experiment. For each condition, the total number of matches (n) is indicated in the figure. Chi-square test was used to determine whether males of a given genetic manipulation respond to amino acids (left) or capsaicin (right). Blue bar indicates average copulation percentage, and the dashed line indicates chance level. AAs, amino acids (200 mM). S, sucrose (50 mM).
(a–b) Optogenetic activation of gut Dh31+ enteroendocrine cells promotes courtship and suppresses protein feeding. After a brief optogenetic stimulation of gut Dh31+ cells, 24-h starved males directly engage in courtship, unlike control flies that typically consumed amino acids (200 mM) before proceeding to courtship. Male flies were exposed to 660-nm LED light for 5 min and then transferred to the behavioral chamber for video recording for 30 min. Flies: Gut Dh31-GAL4: Dh31-GAL4, Tsh-GAL80. UAS: UAS-CsChrimson. Gut Dh31 CsChrimson flies contain all three transgenes. (a) Raster plots showing second-by-second behaviors of 5 representative males of each genotype. Behaviors: feeding (blue), courtship (orange) and copulation (black). (b) Fraction of time spent by each male on feeding and courtship. One-way ANOVA followed by Tukey’s test. n = 7 flies for each condition and genotype. (c–f) Dh31 in the gut and Dh31R in brain Crz+ neurons are required for males to transition from feeding to courtship. The Raster Plots show the second-by-second behaviors of 5 representative males. Gut Dh31-GAL4: Dh31-GAL4, Tsh-GAL80. Starved, 24 h. (c) Gut Dh31 knockdown reduced courtship activity and increased feeding in males ad libitum fed with standard fly food containing protein and carbohydrates. The fraction of time individual males spent on feeding and courtship is shown. Gut Dh31 KD flies had UAS-Dh31-RNAi and Gut Dh31-GAL4. One-way ANOVA followed by Tukey’s test. n = 7 flies for each condition. (d) Gut Dh31 knockdown increased feeding duration and eliminated courtship when food contained amino acids, but had no effect when food contained only sucrose. (e) Chemogenetic activation of gut Dh31-GAL4 cells. The ingestion of capsaicin-containing food induced courtship but food containing only sucrose had no effect. Gut Dh31 VR1 flies contained UAS-VR1 and Gut Dh31-GAL4. (f) Dh31R knockdown in brain Crz+ neurons eliminated the effect of amino acids on courtship, but not feeding. GAL4: Crz-GAL4, tub>GAL80>stop. UAS: otd-nls::flp, UAS-Dh31R-RNAi. Brain Dh31R KD flies had all four transgenes.
Extended Data Fig. 7 Expression of Dh31R and Crz in brain Crz+ neurons is required for the aphrodisiac effect of amino acids.
(a) Validation of Dh31R knockdown. qRT-PCR results show that Dh31R expression level is reduced in flies containing the Actin-GAL4 and UAS-Dh31R-RNAi transgenes. RNA was extracted from 5 male flies per experiment, with 6 experiments for each genotype. One-way ANOVA followed by Tukey’s test. (b–d) Schematic indicates genetic strategies to perturb Crz+ neurons in the brain and VNC (b), only in the brain (c), and only in the VNC (d). Representative confocal images from 3 samples show the different intersections between Crz-GAL4 and otd-nls::flp (corresponding genotypes are shown below the images). Samples were immuostained with anti-GFP (green), nc82 (blue). Scale bar, 50 μm. The courtship effect of Dh31R knockdown in both brain and VNC, in brain only or in VNC only. Each data point represents one experiment. For each condition, the total number of matches (n) is indicated in the figure. p-value was determined by Chi-square test to indicate whether males of a given genetic manipulation respond to amino acids. Blue bar indicates average copulation percentage, and the dashed line indicates chance level. AAs, amino acids (200 mM). S, sucrose (50 mM). (e) Validation of Crz knockdown. Immunostaining of anti-Crz (red) showed Crz is undetectable in brain Crz+ neurons in Crz-Gal4, UAS-Crz-RNAi flies, based on 3 independent experiments. Scale bar, 50 µm. (f–g) The courtship effect of Crz knockdown in brain Crz+ neurons (f) or in VNC Crz+ neurons (g). For each condition, the total number of matches (n) from 5 independent experiments is shown in the figure. p-value was determined by Chi-square test to indicate whether males of a given genetic manipulation respond to amino acids. Blue bar indicates average copulation percentage, and the dashed line indicates chance level. AAs, amino acids (200 mM). S, sucrose (50 mM).
(a) Response of Dh31+ cells to different nutrients at the indicated concentrations. Suc: sucrose, Fru+Glu: mixture of fructose and glucose, EAA: essential amino acids, NEAA: nonessential amino acids. The average calcium response (∆F/F) is shown in black with SEM as gray shaded areas (n = 6 male flies). (b) The response of individual Dh31+ cells to a mixture of amino acids at different concentrations (amino acid composition is detailed in Supplementary Tables 4). Gray lines below the traces indicate the stimulation period of amino acids or saline control.
Brain Crz+ neurons exhibited increased calcium activity in response to synthetic Dh31 peptide. The evoked response latency decreased with increasing concentration of Dh31, while the peak ∆F/F did not change. Ex vivo brain preparations of flies carrying Crz-GAL4 and UAS-GCaMP7s were imaged with two-photon microscopy. (a) Representative calcium responses (ΔF/F) of brain Crz+ neurons in response to different concentrations of Dh31 peptide. The time of Dh31 application is marked by an arrowhead below the calcium response (ΔF/F) trace. (b) Response latency (left) and peak ΔF/F (right) of brain Crz+ neurons in response to different concentrations of Dh31 peptide. Error bars, SEM. n = 7 samples. Significant differences (p < 0.05) are indicated by different letters, One-way ANOVA followed by Tukey’s test. Detailed statistical analysis is available in Supplementary Tables 7.
(a–b) Effect of CO2 exposure on heartbeat and the response of brain Crz+ neurons to the activation of gut Dh31+ cells. (a) Images show the changing diameter of the heart-tube before, during and after CO2 exposure. Heartbeat frequency was extracted from the changing diameter in male flies expressing tdTomato in cardiomyocytes. The light and dark blue lines show the heartbeat frequency of individual flies and the average, respectively (n = 6 flies). (b) The effect of CO2-exposure on the peak ∆F/F of Crz+ neurons in response to optogenetic activation of Dh31+ enteroendocrine cells. Average ∆F/F traces are shown as solid lines with SEM as shaded areas. Stimulation intensity: 1.75 mW/mm2. Error bars, SEM. Two-tailed t-test (n = 5 samples). (c) Schematic of a three-photon microscope, custom built with a MOM scope (Sutter). Pump laser emission of 1,035 nm is converted to 1,320 nm or 1,650 nm with OPA. Average laser power was controlled by a half-wave plate (λ/2) and a polarizing beamsplitter (BS). The pulse width of 1,320 nm emission was compressed through the light path containing P1, P2, RM, Mpu, KT1 and the pulse width of 1,650 nm emission was compressed by the light path containing M1, Si, M2. Beam size at the scan mirror (SM) was matched by KT2. Green and Red emission was collected from the sample with two photomultiplier tubes (PMTs). LP: 1200 nm long pass filter; MM: movable mirror; Mpu: pick up mirror; P1, P2: prism; RM: roof mirror; KT1, KT2: keplerian telescope; Si: silicon crystal; M1, M2: mirror; SL: scan lens; TL: tube lens.
Extended Data Fig. 11 Identification of brain Dh31R+ neurons that regulate the intake of protein-containing food.
(a) Expression pattern of neuropeptides, neuropeptidergic driver lines and Dh31R in the brain. Top row: arrowheads indicate neurons that co-express Dh31R with AstA-GAL4, Lk-GAL4, or Dsk-GAL4. Bottom row: arrowheads indicate neurons that co-express AstC with Dh31R or R67F03-GAL4. Scale bar, 50 μm. (b) Food intake of flies with Dh31R knockdown in different neurons. Gal4 lines (Itp, AstA, LK, R67F03) were crossed to UAS-Dh31R-RNAi and tested for amino acid food consumption. Male flies with Dh31R knockdown in R67F03-GAL4 cells consumed more amino acids than controls. The number of independent experiments is indicated in each panel. One-way ANOVA followed by Tukey’s test. (c and d) Genetic strategies to label R67F03-GAL4 neurons in the brain (c) and outside the brain (d). Representative confocal images from 3 independent experiments show the different intersections between R67F03-GAL4 and otd-nls::flp (corresponding genotypes are shown). Samples were immunostained with anti-GFP (green), nc82 (blue). Scale bar, 50 μm. (e) Knockdown of Dh31R (left panel) or AstC (right panel) in R67F03-GAL4 cells outside the brain. Knockdown male flies consumed the same amount of amino acids and sucrose food compared with control flies. GAL4: R67F03-GAL4, otd-nls::flp; UAS: UAS-Dh31R-RNAi, tub>stop>GAL80; Dh31R KD flies have all four transgenes (left panel). GAL4: R67F03-GAL4, otd-nls::flp; UAS: UAS-AstC-RNAi, tub>stop>GAL80; AstC KD flies have all four transgenes (right panel). n = 10 independent experiments for each condition/genotype. One-way ANOVA test. (f–g) The elevated calcium levels in brain AstC+ neurons in response to dietary amino acids require Dh31R expression. Calcium levels in brain AstC+ neurons were higher in male flies fed with amino acids compared to those fed with sucrose, as measured by the calcium level reporter, CaLexA. Expression of Dh31R in brain AstC+ neurons was required for the increased calcium levels. Foods: sucrose (50 mM); AAs, amino acids (200 mM). Flies: Control (R67F03-GAL4, UAS-CaLexA), Dh31R KD (R67F03-GAL4, UAS-CaLexA, UAS-Dh31R RNAi). Confocal images show representative samples. Scale bar, 20 μm. Box plots show quantification of fluorescence intensity from AstC+ neurons. 2 neurons per sample. The number of samples for each condition is indicated. Two-tailed Mann-Whitney U test.
(a–d) Courtship and protein feeding behavior of flies expressing TrpA1 in brain Crz+ or AstC+ neurons at 21 °C. (a, b) The copulation percentages and total protein consumption (200 mM AAs) of male flies expressing TrpA1 in brain Crz+ neurons and controls. Flies: Bain Crz-GAL4, otd-nls::flp; UAS: UAS>stop>TrpA1; Brain Crz TrpA1 flies have all three transgenes. (c, d) The copulation percentages and total protein consumption (200 mM AAs) of male flies expressing TrpA1 in brain AstC+ neurons and controls. Flies: Brain Astc-GAL4: R67F03-GAL4, otd-nls::flp; UAS: UAS>stop>TrpA1; Brain AstC TrpA1 flies contains all three transgenes. (e) AstC knockdown in brain AstC+ neurons does not affect male courtship. Male flies with AstC knockdown in brain AstC+ neurons exhibited similar copulation percentage as their genetic controls. Three males of different genotypes were given access to one virgin female in three-male courtship assay. Copulation percentages of the different genotypes in the three-male courtship assay are shown, with the average value of each genotype indicated by a horizontal bar. Lines connect results from the same experiment (a, c, e). The number of independent experiments is indicated in each panel. One-way ANOVA test.
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Lin, HH., Kuang, M.C., Hossain, I. et al. A nutrient-specific gut hormone arbitrates between courtship and feeding. Nature 602, 632–638 (2022). https://doi.org/10.1038/s41586-022-04408-7
Signal Transduction and Targeted Therapy (2022)
Neuroscience Bulletin (2022)