Molecular characterization of gustatory second-order neurons reveals integrative mechanisms of gustatory and metabolic information
- Instituto de Neurociencias, CSIC-UMH, Spain
Figures
Figure 1 with 2 supplements
The metabolic state affects the gene profiles of the Gustatory Second-Order Neurons (G2Ns) populations analyzed.
(A) The scheme depicts the region of the brain dissected and the sorting of the G2Ns according to the input. Two populations of trans-Tango neurons were labeled using Gr64f-Gal4 and Gr66a-Gal4 transgenic lines. Flies were either fed or food-starved for 24 hr. (B) Principal Component Analysis (PCA) of the genes. (C) Volcano plots show the genes up- and down-regulated in fed and starved conditions in the two populations of G2Ns.
Figure 1—figure supplement 1
Variation in Gustatory Second-Order Neuron (G2N) numbers for the different populations under study.
Immunofluorescence with anti-GFP (green), anti-RFP (magenta), and anti-nc82 (blue) on a whole-mount brain on a (A) Gr64f-Gal4>trans-Tango and (B) Gr66-Gal4>trans-Tango. Quantification of the number of G2Ns for each population, (C) Gr64fG2Ns (n=11 brains) and (D) Gr66aG2Ns (n=9 brains). Larvae were grown at 25 °C and 21 d at 18 °C after pupation. Scale bar = 50 μm.
Figure 1—figure supplement 2
Gene Ontology (GO) analysis of the genes differentially expressed in fed and starved conditions for the two Gustatory Second-Order Neurons (G2Ns) populations analyzed.
Graphical representation of the GO terms obtained from the Gene Set Enrichment Analysis (GSEA), grouped by the GO category. The data represent the fold enrichment with a heatmap, and the number of genes included in each GO term by size for all G2Ns and metabolic states analyzed.
Figure 2
Transcripts per Million (TPM) for Neuropeptides and Neurotransmitters.
TPMs for Neurotransmitters (A), Neurotransmitter Receptors (B), Neuropeptides (C), and Neuropeptide Receptors (D) for the two Gustatory Second-Order Neuron (G2N) populations in the two metabolic conditions studied (fed and 24 hr starved).
Figure 3
Leucokinin expression is increased in starvation.
(A) Immunofluorescence with anti-GFP (green) and anti-nc82 (blue) on a whole-mount brain of a Lk-Gal4 >UAS-mCD8::GFP. (B) Scheme showing the approach used to select the Lk GFP+ neurons by Fluorescent Activated Cell Sorting (FACS). (C) qPCR results for Lk neurons labeled with GFP and sorted by FACS. The central brain includes four Lk neurons, while the SEZ region includes only the two neurons located in this region. (D) Intensity fluorescence quantification in whole Oregon-R brains stained with anti-Lk antibody in fed and 24 hr starved flies. SELK Fed = 20 brains; SELK Starved = 21 brains; LHLK Fed = 12 brains; LHLK Starved = 16 brains. Statistical test: Wilcoxon rank sum test. *p<0.05, **p<0.01, ****p<0.0001. Scale bar = 50 μm.
Figure 4
Lk neurons co-localize with the trans-Tango signal.
Immunofluorescence with anti-Leucokinin (blue) and anti-RFP (magenta) on whole-mount brains of (A) Gr64f-Gal4>trans-Tango, and (B) Gr66a-Gal4>trans-Tango. Images correspond to the adult Subesophageal Zone (SEZ). Arrowheads point to SELK neurons. Scale bar = 50 μm.
Figure 5
Lk neurons are synaptically connected to Gr64fGRNs and Gr66aGRNs.
(A) GRASP: Immunofluorescence with anti-GFP (green) and anti-N-cadherin (blue) on whole-mount brains of Gr64f-LexA>LexAop-CD4spGFP (Scott, 2018) and Lk-Gal4 >UAS-CD4spGFP (Chu et al., 2014; Inagaki et al., 2014; LeDue et al., 2016; Yarmolinsky et al., 2016; Vosshall and Stocker, 2007; Dahanukar et al., 2007; Fujii et al., 2015; Dunipace et al., 2001; Wang et al., 2004 flies). (B) Active-GRASP: Immunofluorescence with anti-GFP (green) and anti-N-cadherin (blue) on whole-mount brains of Gr64f-LexA>LexAop-nSybspGFP (Chu et al., 2014; Inagaki et al., 2014; LeDue et al., 2016; Yarmolinsky et al., 2016; Vosshall and Stocker, 2007; Dahanukar et al., 2007; Fujii et al., 2015; Dunipace et al., 2001; Wang et al., 2004) and Lk-Gal4 >UAS-CD4spGFP (Scott, 2018) stimulated with water (left column) and sucrose (right column). (C) GRASP: Immunofluorescence with anti-GFP (green) and anti-N-cadherin (blue) on whole-mount brains of Gr66a-LexA>LexAop-CD4spGFP (Scott, 2018) and Lk-Gal4 >UAS-CD4spGFP (Chu et al., 2014; Inagaki et al., 2014; LeDue et al., 2016; Yarmolinsky et al., 2016; Vosshall and Stocker, 2007; Dahanukar et al., 2007; Fujii et al., 2015; Dunipace et al., 2001; Wang et al., 2004). (D) Active-GRASP: Immunofluorescence with anti-GFP (green) and anti-N-cadherin (blue) on whole-mount brains of Gr66a-LexA>LexAop-nSybspGFP (Chu et al., 2014; Inagaki et al., 2014; LeDue et al., 2016; Yarmolinsky et al., 2016; Vosshall and Stocker, 2007; Dahanukar et al., 2007; Fujii et al., 2015; Dunipace et al., 2001; Wang et al., 2004) and Lk-Gal4 >UAS-CD4spGFP (Scott, 2018) stimulated with water (left column) and caffeine (right column). (E) Immunofluorescence with anti-GFP (green), anti-RFP (magenta), and anti-nc82 (blue) on whole-mount brains of Gr64f-LexA, Lk-Gal4 >BacTrace (top row) and Gr66a-LexA, Lk-Gal4 >BacTrace (bottom row). Arrowheads point to the BacTrace positive signal. Scale bar = 50 μm.
Figure 6 with 1 supplement
Lk neurons are pre- and postsynaptic to a significant number of neurons.
(A) The anatomy of the SELK neurons (Right in green and Left in blue) was reconstructed using the FAFB brainmesh template in Flywire. IDs from Flywire are indicated: 720575940629543409 ID for the Left SELK in blue (GNG.595/DNg68(L)) and 720575940630808827 for the Right SELK in green (GNG.685/DNg68(R)). Zoom in to the Subesophageal Zone (SEZ) from (A), showing the details of the neuronal arborizations. (B) SELK postsynaptic candidate neurons reconstructed in the FAFB (Full Adult Fly Brain) brainmesh template in Flywire. Only postsynaptic candidate neurons with ≥10 synaptic points with SELK neurons are shown (57 postsynaptic candidate neurons). (C) Immunofluorescence with anti-GFP (green), anti-RFP (magenta), and anti-nc82 (blue) on a whole-mount brain of a Lk-Gal4 >trans-Tango flies. (D) SELK presynaptic candidate neurons reconstructed in the FAFB brain mesh template in Flywire. Only presynaptic candidate neurons with ≥10 synaptic points with SELK neurons are shown (101 presynaptic candidate neurons). (E) Immunofluorescence with anti-GFP (green), anti-RFP (magenta), and anti-nc82 (blue) on a whole-mount brain of a Lk-Gal4 >retro-Tango fly. Scale bar: 50 µm.
Figure 6—figure supplement 1
Sequential methodology to identify a strong SELK candidate neuron in the FAFB connectome.
(A) Reconstruction representation in the Full Adult Fly Brain (FAFB) brainmesh template in Flywire from all groups of Gustatory Receptor Neurons (GRNs) analyzed, both bitter (group 1: orange and group 2: yellow) and sweet (group 4: green and group 5: blue) GRNs. (B) Top postsynaptic neurons to bitter and sweet GRNs were analyzed and considered as top SELK candidate neurons based on visual comparisons. (C) Alignment of the right SELK neuron segmented skeleton in the FAFB by using the Flywire Gateway app. (D) Top right SELK candidate neuron based on localization and morphology similarities with the right SELK neuron alignment from (C). ID from Flywire is indicated in green: 720575940630808827 (GNG.685/DNg68(R)). Zoom in on the Subesophageal Zone (SEZ) from (D), showing detailed similarities of the right SELK candidate neuron arborization with the right SELK skeleton alignment, both in front and side view. Scale bar: 50 μm.
Figure 7 with 1 supplement
Leucokinin neurons integrate sweet and bitter gustatory information.
(A) Scheme depicting the experimental procedure for the Proboscis Extension Reflex (PER). Flies are given a series of increasing concentrations of sucrose (6.25, 12.5, 25, 50, 100, 200, 400, and 800 mM) for Experiment 1; and a fixed concentration of sucrose (50 mM) mixed with increasing concentrations of caffeine (1, 5, 10, and 20 mM) for Experiment 2. All flies starved 24 hr. (B) PER dose response curve of Lk-Gal4 >UAS-TNTimp (n=30) and Lk-Gal4 >UAS TNT (n=30) flies for Experiment 1. (C) PER of Lk-Gal4 >UAS-TNTimp (n=30) and Lk-Gal4 >UAS TNT (n=30) flies for Experiment 2. (D) Scheme depicting the experimental procedure for flyPAD (Adapted from Itskov et al., 2014). Experiment 3: 20 mM Sucrose vs. 100 mM Sucrose and Experiment 4: 20 mM Sucrose vs. 100 mM Sucrose +50 mM Caffeine. All flies starved for 24 hr. (E) Preference Index (Left) for the highest concentration of sucrose (100 mM) with or without 50 mM caffeine and total number of sips (Right) for Experiments 3 and 4. Experiment 3: Lk-Gal4 >UAS-TNTimp (n=48); Lk-Gal4 >UAS TNT (n=59); Experiment 4: Lk-Gal4 >UAS-TNTimp (n=20); Lk-Gal4 >UAS TNT (n=25). Only flies that performed a minimum of 25 sips per fly and two bouts were considered for the analysis. ns: non-significant, *p<0.05. PER analysis: Logistic regression model with binomial distribution; Error bars represent the standard error of the proportion. flyPAD statistics Wilcoxon Rank Sum Test.
Figure 7—figure supplement 1
Leucokinin neurons are essential to discriminate sweet and bitter stimuli.
(A) PER of Lk-Gal4 >tsh-Gal80, UAS-TNTimp (n=30) and Lk-Gal4 >tsh-Gal80, UAS-TNT (n=30) for a mixture of 50 mM Sucrose with increasing concentrations of caffeine. (B) PER of Lk-Gal4/+, UAS-RNAiLk/+and Lk-Gal4 >UAS-RNAiLk (n=30), for a mixture of 100 mM Sucrose with increasing concentrations of caffeine. *p<0.05, **p<0.01. Logistic regression model with binomial distribution. Error bars represent the standard error of the proportion.
Figure 8
Model for the integration of gustatory information by SELK neurons.
(A) SELK neurons receive direct input from Gr64fGRNs (sweet), Gr66aGRNs (bitter), and metabolic information. When flies are fed, the expression of the neuropeptide Leucokinin is reduced. The probability of a fed fly initiating feeding is reduced when facing any food. (B) SELK neurons in starved flies express large amounts of Leucokinin. In this situation, flies are more prone to accept food containing bitter compounds. (C) Starved flies with silenced SELK neurons are less prone to accept sweet foods laced with bitter compounds.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (D. melanogaster) | Oregon-R | Kindly donated by Prof. Richard Benton | NA | |
| Strain (D. melanogaster) | w1118 | Kindly donated by Prof. Francisco Tejedor | NA | |
| Strain (D. melanogaster) | Gr64f-Gal4 | Kindly donated by Prof. Richard Benton | NA | |
| Genetic reagent (D. melanogaster) | Gr64f-LexA | Kindly donated by Prof. Richard Benton | NA | |
| Genetic reagent (D. melanogaster) | Gr66a-Gal4 | Bloomington Drosophila Stock Center | BDSC 57670 | |
| Genetic reagent (D. melanogaster) | Gr66a-LexA; LexAop-rCD2::GFP | Kindly donated by Prof. Richard Benton | NA | |
| Genetic reagent (D. melanogaster) | Lk-Gal4 (2nd Chr) | Bloomington Drosophila Stock Center | BDSC 51993 | |
| Genetic reagent (D. melanogaster) | Lk-Gal4 (X Chr) used only in the tsh-Gal80 experiment | Bloomington Drosophila Stock Center | BDSC 51992 | |
| Genetic reagent (D. melanogaster) | retro-Tango (QUAS-mtdTomato; retro-Tango; UAS-retro-Tango-GFP) | Bloomington Drosophila Stock Center | BDSC 99661 | |
| Genetic reagent (D. melanogaster) | UAS-mCD8::GFP | Bloomington Drosophila Stock Center | BDSC 32186 | |
| Genetic reagent (D. melanogaster) | trans-Tango (UAS-myrGFP, QUAS-mtdTomato; trans-Tango) | Bloomington Drosophila Stock Center | BDSC 77124 | |
| Genetic reagent (D. melanogaster) | UAS-TNT | Bloomington Drosophila Stock Center | BDSC 28837 | |
| Genetic reagent (D. melanogaster) | UAS-TNTimp | Bloomington Drosophila Stock Center | BDSC 28839 | |
| Genetic reagent (D. melanogaster) | UAS-RNAiLk | Bloomington Drosophila Stock Center | BDSC 25798 | |
| Genetic reagent (D. melanogaster) | GRASP (UAS-CD4-spGFP1-10, lexAop-CD4-spGFP11) | Bloomington Drosophila Stock Center | BDSC 58755 | |
| Genetic reagent (D. melanogaster) | Active GRASP (lexAop-nSyb-spGFP1-10, UAS-CD4-spGFP11; MKRS/TM6B) | Bloomington Drosophila Stock Center | BDSC 64315 | |
| Genetic reagent (D. melanogaster) | BAcTrace (LexAop-GFP, LexAop-QF; UAS-B3RT, QUAS-mtdTomato) | Bloomington Drosophila Stock Center | BDSC 90826 | |
| Genetic reagent (D. melanogaster) | tsh-Gal80 | Kindly donated by Prof. María Domínguez | ||
| Antibody | GFP (Green Fluorescent Protein) | Abcam | ab13970 | 1:500 |
| Antibody | RFP (Red Fluorescent Protein) | Abcam | ab62341 | 1:500 |
| Antibody | nc82 Bruchpilot | DSHB | 1:50 | |
| Antibody | GFP reconstructed in GRASP | Sigma-Aldrich | G6539 | 1:500 |
| Antibody | Leucokinin | Kindly donated by Prof. Pilar Herrero | 1:100 | |
| Antibody | Cadherin DN-Extracellular Domain | Jackson Inmunoresearch | 112-165-003 | 1:500 |
| Antibody | Anti-Chicken Alexa 488 | Abcam | ab150169 | 1:250 |
| Antibody | Anti-Rabbit Cy3 | Jackson Inmunoresearch | 111-165-144 | 1:250 |
| Antibody | Anti-Mouse Cy5 | Jackson Inmunoresearch | 115-175-146 | 1:250 |
| Antibody | Anti-Mouse Cy3 | Jackson Inmunoresearch | 115-165-166 | 1:250 |
| Antibody | Anti-Mouse Alexa 488 | Jackson Inmunoresearch | 115-545-003 | 1:250 |
| Antibody | Anti-Rat Cy3 | Jackson Inmunoresearch | 112-165-167 | 1:250 |
| Antibody | Anti-Rat Alexa 697 | Jackson Inmunoresearch | 112-605-167 | 1:250 |
| Chemical compound | Sucrose | Sigma Aldrich | 102174662 | |
| Chemical compound | Caffeine | Sigma Aldrich | 102143502 | |
| Chemical compound | Agarose | Condalab | 8100.00 | |
| Chemical compound | Agarose (low melting) | Lonza | 50100 |
Additional files
-
Supplementary file 1
Number of brains dissected and cells sorted for the RNAseq experiment.
- https://cdn.elifesciences.org/articles/100947/elife-100947-supp1-v1.xlsx
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Supplementary file 2
List of candidate IDs.
- https://cdn.elifesciences.org/articles/100947/elife-100947-supp2-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/100947/elife-100947-mdarchecklist1-v1.pdf
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Molecular characterization of gustatory second-order neurons reveals integrative mechanisms of gustatory and metabolic information
eLife 13:RP100947.
https://doi.org/10.7554/eLife.100947.3
