Taste quality and hunger interactions in a feeding sensorimotor circuit
- University of California, Berkeley, United States
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- Queensland Brain Institute, University of Queensland, Australia
Figures
Figure 1 with 2 supplements
Sugar-sensing gustatory receptor neurons (GRNs) synapse onto multiple second-order neurons that influence proboscis extension.
(A) Aggregate synaptic connectivity from sugar GRNs onto second-order sugar neurons. Numbers indicate the total number of synapses that the 17 candidate sugar GRNs make onto each second-order neuron. (B–C) Manually reconstructed electron microscopy (EM) skeletons (B) and registered neural images in split-Gal4 lines (C) for each second-order neuron in the subesophageal zone (SEZ) of the Drosophila brain. Sugar GRNs are depicted in white, JRC 2018 unisex coordinate space is shown in gray (C). Scale bar is 50 μm. (D) CsChrimson-mediated activation of seven second-order neurons elicits proboscis extension, n=30 flies per genotype. (E) GtACR1-mediated inhibition of second-order neurons reduces proboscis extension to 50 mM sucrose, n=46–83 flies per genotype. (D–E) The fraction of flies exhibiting proboscis extension response (PER) upon optogenetic or 50 mM sucrose stimulation. Mean ± 95% confidence interval (CI), Fisher’s Exact Tests, *p<0.05, ***p<0.001. See Figure 1—figure supplement 1 for EM reconstructions of additional second-order neurons and synaptic connectivity counts. See Figure 1—figure supplement 2 for additional PER phenotypes of second-order sugar neurons.
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Figure 1—source data 1
Source data for behavioral experiments in Figure 1.
- https://cdn.elifesciences.org/articles/79887/elife-79887-fig1-data1-v3.xlsx
Figure 1—figure supplement 1
Anatomy of reconstructed second-order neurons and their connectivity, related to Figure 1.
(A) Schematic of the entire fly brain, showing electron microscopy (EM) reconstructed sugar-sensing gustatory receptor neurons (GRNs) in gray. Full adult fly brain (FAFB) neuropil space is shown in darker gray. Area outlined in red is enlarged in the first panel of C. (B) Synaptic connectivity of 17 previously identified candidate sugar GRNs onto second-order neurons that elicit proboscis extension response (PER). Line thickness represents the number of synapses, with a minimum of six synapses to a maximum of 46 synapses. (C) Anatomy of second-order candidate sugar EM reconstructed neurons. Scale bar is 50 μm. (D) Synaptic connectivity from sugar GRNs onto and from second-order neurons. Second-order neurons identified by EM and present in a split-Gal4 line (black circles); second-order neurons identified by EM only (gray circles). (E) Synaptic connectivity between second-order neurons. (F) Neurons with the most synapses from 17 candidate sugar GRNs based on Flywire predicted synapses (n≥40), with x-axis labeling neurons identified in this study.
Figure 1—figure supplement 2
Additional proboscis extension phenotypes of second-order neurons, related to Figure 1.
(A) Light microscopy images of Cleaver, Usnea, and Fudog. Specific lines for Cleaver and Usnea were generated using a triple-intersection approach (see Methods). In the Fudog image, sugar GRNs are depicted in white. Scale bar is 50 μm. JRC 2018 unisex coordinate space is shown in blue (Cleaver and Usnea) or dark gray (Fudog). (B) Activation of Usnea, but not Cleaver or Fudog, elicits proboscis extension. n=30 flies per genotype. (C) Hyperpolarization of Rattle and Usnea inhibited proboscis extension to 100 mM sucrose, but hyperpolarization of other second-order neurons did not. n=30-144 flies per genotype. (B-C) Mean ± 95% confidence interval (CI), Fisher’s Exact Tests, ***p<0.001.
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Figure 1—figure supplement 2—source data 1
Source data for behavioral experiments in Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/79887/elife-79887-fig1-figsupp2-data1-v3.xlsx
Figure 2 with 1 supplement
Second-order neurons synapse onto a local sensorimotor circuit for feeding initiation.
(A) Schematic of the feeding initiation circuit. Circles outlined in black denote neurons with split-Gal4 genetic access, circles with gray outlines denote neurons without split-Gal4 genetic access. Line thickness represents synaptic connectivity of more than five synapses. (B–C) Electron microscopy (EM) neural reconstructions (B) and registered neural images in split-Gal4 lines (C) of third-order or premotor neurons in the subesophageal zone (SEZ). Scale bar is 50 μm. JRC 2018 unisex coordinate space is shown in gray, MN9 morphology is shown in orange. (D) CsChrimson-mediated activation of third-order or premotor neurons elicits proboscis extension response (PER), n=30 flies per genotype. (E) GtACR1-mediated inhibition of third-order or premotor neurons does not influence PER to 50 mM sucrose, n=40–70 flies per genotype. (D–E) Mean ± 95% CI, Fisher’s Exact Tests, ***p<0.001. See Figure 2—figure supplement 1 for synaptic counts.
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Figure 2—source data 1
Source data for behavioral experiments in Figure 2.
- https://cdn.elifesciences.org/articles/79887/elife-79887-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Synaptic connectivity in the feeding initiation circuit, related to Figure 2.
(A) Schematic of the feeding initiation circuit, with circles outlined in black for neurons with split-Gal4 lines, circles outlined in gray for neurons without split-Gal4 lines. Line thickness represents connectivity of more than five synapses, with synapse numbers labeled. (B) Neurons with the most synapses from second-order neurons that elicit proboscis extension response (PER), based on Flywire predicted synapses (n≥30), x-axis labels neurons identified in this study. (C) Neurons with the most synapses onto MN9, based on Flywire predicted synapses (n≥30), x-axis labels neurons identified in this study. (D) Neurotransmitter predictions (Eckstein et al., 2020) for individual synapses for each neuron in the feeding initiation circuit generated by a machine learning classifier. The fraction of synapses predicted to contain each neurotransmitter is indicated by color.
Figure 3 with 1 supplement
Feeding initiation neurons respond to taste detection.
(A) Connectivity schematic of the feeding initiation circuit, where filled green circles represent cell types that respond to sugar detection, while filled blue circles represent cell types that respond to water detection. One cell type, Phantom, responds to both sugar and water (split blue and green circle). Fdg did not respond to proboscis taste detection (white circle), but see Figure S4A for responses to optogenetic activation of sugar gustatory receptor neurons (GRNs). (B) Calcium responses of feeding initiation neurons to stimulation of the proboscis in food-deprived flies. For each cell type, GCaMP6s fluorescence traces are shown on the left of the panel (ΔF/F), while ΔF/F area for each trace is shown on the right, with thin black lines indicating sample pairing. The proboscis of each tested individual was stimulated with water (green), sugar (blue), and bitter (red) tastants in sequential trials during the indicated period (thick black line). The following split-GAL4 lines were imaged for each cell type: Clavicle; SS48947, FMIn; SS48944, Zorro; SS67405, G2N-1; SS47082, Usnea; SS37122, Phantom; SS68204, Rattle; SS50091, Fdg; SS31345, Bract; SS31386, Roundup; SS47744. n=5-8 flies per genotype. Quade’s test with Quade’s All Pairs test, using Holm’s correction to adjust for multiple comparisons, ns p>0.05, *p≤0.05, **p≤0.01. See also Figure 3—figure supplement 1 for additional calcium imaging studies of feeding initiation neurons.
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Figure 3—source data 1
Source data for calcium experiments in Figure 3.
- https://cdn.elifesciences.org/articles/79887/elife-79887-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Taste responses of feeding initiation neurons, related to Figure 3.
(A) Calcium responses of Fdg to optogenetic activation of water (blue, ppk28-LexA), sugar (green, Gr5a-LexA), or bitter (red, Gr66a-LexA) GRNs in food-deprived flies. To examine Fdg responses, GCaMP7b was expressed using SS46913. Fluorescence traces are shown on the left of the panel (ΔF/F), while ΔF/F area for each trace is shown on the right. Stimulation with 660 nm light is indicated with vertical gray bars. Kruskal Wallace test with Dunn’s test using Holm’s correction to adjust for multiple comparisons, n=5-7 flies per genotype, ns p>0.05, *p≤0.05, **p≤0.01. (B–C) Calcium responses of water gustatory sensory neurons and feeding initiation neurons to taste stimulation of the proboscis. GCaMP6s fluorescence traces are shown on the left of each panel (ΔF/F), while ΔF/F area for each trace is shown on the right. n=5-10 flies per genotype, ns p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. (B) Taste responses to water in fed (purple) versus 2 hr desiccated (2 hr dess, teal) flies (left). Taste responses to water in fed flies allowed to rest for 1 hr in low osmolality artificial hemolymph after dissection before imaging (1 hr fed, purple) versus pseudodessicated, thirsty-like flies (p-dess, blue) (right). (C) Taste responses in pseudodessicated, thirsty-like flies. The proboscis of each tested individual was stimulated with water (green), sugar (blue), and bitter (red) tastants in sequential trials during the indicated period (thick black line). Thin black lines indicate sample pairing. The following split-GAL4 lines were imaged for each cell type: Clavicle; SS48947, G2N-1; SS47082, Usnea; SS37122, Rattle; SS50091, Roundup; SS47744. Quade’s test with Quade’s All Pairs test, using Holm’s correction to adjust for multiple comparisons. (D) GRN synapes onto second-order neurons, with GRN categories based on Engert et al., 2021.
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Figure 3—figure supplement 1—source data 1
Source data for calcium imaging experiments in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/79887/elife-79887-fig3-figsupp1-data1-v3.xlsx
Figure 4
Hunger acts on a subset of second-order central neurons to modulate behavior.
(A) Schematic of the feeding initiation circuit, with filled green circles representing nodes that are hunger-modulated. (B) Optogenetic activation at four different light intensities. (C) Activation of sugar-sensing neurons results in different feeding initiation rates between fed and food-deprived flies (left) whereas activation of MN9 does not (right), at four different light intensities. n=50. (D) Optogenetic activation of second-order, third-order, and premotor neurons in either fed or food-deprived flies. n=39–103. Mean ± 95% CI, Fisher’s Exact Tests, ***p<0.001.
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Figure 4—source data 1
Source data for behavioral experiments in Figure 4.
- https://cdn.elifesciences.org/articles/79887/elife-79887-fig4-data1-v3.xlsx
Figure 5 with 1 supplement
Premotor neurons integrate sweet and bitter taste detection.
(A) Schematic of the feeding initiation circuit, showing a pathway from bitter GRNs to premotor neurons. Filled maize circle labels a premotor neuron inhibited by bitter tastants, filled gray circle labels an upstream second-order neuron that is not inhibited by bitter tastants. (B and C) Calcium responses of feeding circuit neurons to optogenetic activation of sugar (green, Gr5a-LexA), sugar plus bitter (maize, Gr5a-LexA plus Gr66a-LexA), or bitter (red, Gr66a-LexA) GRNs in food-deprived flies. For each cell type, Syt::GCaMP7b fluorescence traces are shown on the left of the panel (ΔF/F), while ΔF/F area for each trace is shown on the right. Periods of stimulation with 660 nm light are indicated with vertical gray bars. (B) SS47744 was imaged to examine Roundup responses. (C) SS47082 was imaged to examine G2N-1 responses. (B-C) Kruskal Wallace test with Dunn’s test using Holm’s correction to adjust for multiple comparisons, n=6-8 flies per genotype, ns p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001. See Figure 5—figure supplement 1 for synaptic counts of second-order bitter neurons.
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Figure 5—source data 1
Source data for calcium imaging experiments in Figure 5.
- https://cdn.elifesciences.org/articles/79887/elife-79887-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
Second-order bitter neurons, related to Figure 5.
Neurons with the most synapses from candidate bitter gustatory receptor neurons (GRNs) based on Flywire predicted synapses (n≥30), with x-axis labeling neurons identified in this study.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-Brp (mouse monoclonal) | DSHB, University of Iowa, USA | DSHB Cat# nc82, RRID:AB_2314866 | 1/500 |
| Antibody | Anti-GFP (chicken polyclonal) | Thermo Fisher Scientific | Thermo Fisher Scientific Cat# A10262, RRID:AB_2534023 | 1/1000 |
| Antibody | Anti-dsRed (rabbit polyclonal) | Takara | Takara Bio Cat# 632496, RRID:AB_10013483 | 1/1000 |
| Antibody | Anti-chicken Alexa Fluor 488 (goat polyclonal) | Thermo Fisher Scientific | Thermo Fisher Scientific Cat# A-11039, RRID:AB_2534096 | 1/100 |
| Antibody | Anti-rabbit Alexa Fluor 568 (goat polyclonal) | Thermo Fisher Scientific | Thermo Fisher Scientific Cat# A-11036, RRID:AB_10563566 | 1/100 |
| Antibody | Anti-mouse Alexa Fluor 647 (goat polyclonal) | Thermo Fisher Scientific | Thermo Fisher Scientific Cat# A-21236, RRID:AB_2535805 | 1/100 |
| Chemical Compound, drug | All trans-Retinal | MilliporeSigma | Cat # R2500 | |
| Genetic reagent (D. melanogaster) | 20XUAS-IVS-CsChrimson.mVenus attP18 | Bloomington Stock Center; Klapoetke et al., 2014 | RRID:BDSC_55134 | |
| Genetic reagent (D. melanogaster) | UAS-GtACR1.d.EYFP}attP2 | Bloomington Stock Center | RRID:BDSC_92983 | |
| Genetic reagent (D. melanogaster) | Zorro split-GAL4, SS67405 | Janelia Research Campus | Full genotype: w; R12C04-p65ADZp in attP40; VT043788-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | Zorro split-GAL4, SS67406 | Janelia Research Campus | Full genotype: w; R12C04-p65ADZp in attP40; VT020600-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | Clavicle split-GAL4, SS48947 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; VT020732-p65ADZp in attP40; R17G10-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | G2N-1 split-GAL4, SS47082 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; R12C04-p65ADZp in attP40; VT043658-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | G2N-1 split-GAL4, SS56399 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; R12C04-p65ADZp in attP40; VT020839-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | Rattle split-GAL4, SS50091 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; VT006545-p65ADZp in attP40; VT023745-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | Usnea split-Gal4, SS37122 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; VT037525-p65ADZp in attP40; VT033627-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | Usnea and Cleaver split-Gal4, SS31022 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; VT038544-p65ADZp in attP40; VT019345-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | FMIn split-GAL4, SS48944 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; R81E10-p65ADZp in attP40; R17G10-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | FMIn split-GAL4, SS48949 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; R81E10-p65ADZp in attP40; R21H11-ZpGdbd in attP2 | |
| Genetic reagent (D. melanogaster) | Phantom split-GAL4, SS43877 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; R82F02-p65ADZp in attP40; R20G06-ZpGdbd in attP2 | |
| Genetic reagent (D. melanogaster) | Phantom split-GAL4, SS43879 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; R20G06-p65ADZp in attP40; R82F02-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | Phantom split-GAL4, SS68204 | Janelia Research Campus | Full genotype: w; R20G06-p65ADZp in attP40; R81A07-ZpGdbd in attP2 | |
| Genetic reagent (D. melanogaster) | Fudog split-GAL4, SS35290 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; R59F08-p65ADZp in attP40; R69E06-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | Fudog split-GAL4, SS35291 | Janelia Research Campus; Sterne et al., 2021); available at http://splitgal4.janelia.org | Full genotype: w; VT038225-p65ADZp in attP40; R69E06-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | Bract split-GAL4, SS31320 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; R25A01-p65ADZp in attP40; VT058723-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | Bract split-GAL4, SS31386 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; R25A01-p65ADZp in attP40; R37D11-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | Fdg split-GAL4, SS31333 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; R81E10-p65ADZp in attP40; VT037804-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | Fdg split-GAL4, SS46913 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; R81E10-p65ADZp in attP40; R88C07-ZpGdbd in attP2 | |
| Genetic reagent (D. melanogaster) | Roundup split-GAL4, SS47744 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; R23G11-p65ADZp in attP40; VT003236-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | Roundup split-GAL4, SS47745 | Janelia Research Campus; Sterne et al., 2021; available at http://splitgal4.janelia.org | Full genotype: w; R11B11-p65ADZp in attP40; VT003236-ZpGDBD in attP2 | |
| Genetic reagent (D. melanogaster) | 20xUAS >dsFRT > csChrimson-mVenus | Wu et al., 2016 | ||
| Genetic reagent (D. melanogaster) | 8XLexAop2-FLPL(attP40) | Bloomington Stock Center | RRID:BDSC_55820 | |
| Genetic reagent (D. melanogaster) | ;;Dfd-LexA | Simpson, 2016 | ||
| Genetic reagent (D. melanogaster) | ;;Scr-LexA | Simpson, 2016 | ||
| Genetic reagent (D. melanogaster) | w[1118]; 20XUAS-IVS-GCaMP6s(attP40); | Bloomington Stock Center | RRID: BDSC_42746 | |
| Genetic reagent (D. melanogaster) | Gr64f-Gal4 (II) | Kwon et al., 2011 | ||
| Genetic reagent (D. melanogaster) | Ppk28-Gal4 | Bloomington Stock Center; Cameron et al., 2010 | RRID:BDSC_93020 | |
| Genetic reagent (D. melanogaster) | Gr64f-LexA | Miyamoto et al., 2012 | ||
| Genetic reagent (D. melanogaster) | Gr66a-LexA(II) | Thistle et al., 2012; Bloomington Stock Center; | RRID:BDSC_93023 | |
| Genetic reagent (D. melanogaster) | Gr66a-LexA5(III) | Thistle et al., 2012 | ||
| Genetic reagent (D. melanogaster) | UAS-CD8-tdTomato;; | Thistle et al., 2012 | ||
| Genetic reagent (D. melanogaster) | w[1118]; 20XUAS-IVS-GCaMP6s(attP40); | Bloomington Drosophila Stock Center | RRID: BDSC_42746 | |
| Genetic reagent (D. melanogaster) | w[1118];; 20XUAS-IVSGCaMP6s(VK00005) | Bloomington Drosophila Stock Center | RRID: BDSC_42749 | |
| Genetic reagent (D. melanogaster) | Gr5a-LexA-VP16(II) | Gordon and Scott, 2009 | RRID:BDSC_93014 | |
| Genetic reagent (D. melanogaster) | ppk28-LexA(III) | Thistle et al., 2012 | ||
| Genetic reagent (D. melanogaster) | 13XLexAop2-IVS-p10-ChrimsonR-mCherry(attP18) | Vivek Jarayaman | ||
| Genetic reagent (D. melanogaster) | 20XUAS-IVS-jGCaMP7b(attP5) | Bloomington Stock Center | RRID:BDSC_80907 | |
| Genetic reagent (D. melanogaster) | 20XUAS-IVS-jGCaMP7b(VK00005) | Bloomington Stock Center | RRID:BDSC_79029 | |
| Genetic reagent (D. melanogaster) | 20xUAS-IVS-Syn21-Syt::Op-jGCaMP7b(attP18) | Vivek Jarayaman, Chuntao Dan | ||
| Software, Algorithm | Fiji | https://fiji.sc/ | RRID: SCR_002285 | |
| Software, Algorithm | Computational Morphometry Toolkit (CMTK) | Masse et al., 2012 | ||
| Software, Algorithm | NBLAST | Costa et al., 2016; http://nblast.virtualflybrain.org:8080/NBLAST_on-the-fly/; http://flybrain.mrc-lmb.cam.ac.uk/si/nblast/www/ | ||
| Software, Algorithm | VVDviewer | Otsuna et al., 2018; https://github.com/takashi310/VVD_Viewr | ||
| Software, Algorithm | GraphPad Prism | Graphpad Software; https://www.graphpad.com/scientific-software/prism/ | RRID:SCR_002798 | |
| Software, Algorithm | Python | Python Software Foundation; https://www.python.org/downloads/ | ||
| Software, Algorithm | Flywire | Flywire; https://flywire.ai/ | RRID:SCR_019205 | |
| Software, Algorithm | Adobe Illustrator | Adobe Software; https://www.adobe.com/products/illustrator.html | ||
| Software, Algorithm | CATMAID | Saalfeld et al., 2009; https://catmaid.org | ||
| Software, Algorithm | CAVE (connectome annotation versioning engine) | https://github.com/seung-lab/CAVEclient/blob/master/FlyWireSynapseTutorial.ipynb | ||
| Software, Algorithm | R Project for Statistical Computing | R Development Core Team, 2018 | RRID:SCR_001905 | |
| Software, Algorithm | CircuitCatcher | Bushey, 2019; https://github.com/DanBushey/CircuitCatcher | ||
| Software, Algorithm | PMCMRplus package | Pohlert, 2021; https://CRAN.R-project.org/package=PMCMRplus | ||
| Software, Algorithm | SciPy package | Virtanen et al., 2020; https://scipy.org/ | ||
| Software, Algorithm | scikit-posthocs package | Terpilowski, 2018; https://scikit-posthocs.readthedocs.io/en/latest/ |
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Taste quality and hunger interactions in a feeding sensorimotor circuit
eLife 11:e79887.
https://doi.org/10.7554/eLife.79887
