Hugin-AstA circuitry is a novel central energy sensor that directly regulates sweet sensation in Drosophila and mouse
- Institute of Molecular Physiology, Shenzhen Bay Laboratory, China
- Laboratory for Clinical Medicine, School of Public Health, Capital Medical University, China
- Chinese Institutes for Medical Research, China
- Center for Neurointelligence, School of Medicine, Chongqing University, China
- Guangyang Bay Laboratory, Chongqing Institute for Brain and Intelligence, China
Figures
Figure 1 with 8 supplements
hugin+ neurons were a novel glucose sensor in the fly brain.
(A) Experimental procedure schematic. Newly eclosed flies were gathered and provided with regular food for 5 days, followed by a 12 hr period of starvation. Proboscis extension reflex (PER) assays were conducted. (B) Fraction of flies showing PER to different concentrations of sucrose (two-way ANOVA; *, p=0.0349; ***, p=0.0002; ****, p<0.0001; n=4 groups, each of 10 flies). (C–D) Fraction of flies of the indicated genotypes under 30°C showing PER to 400 mM of sucrose (one-way ANOVA; *, p=0.0233; **, p=0.0054; ***, p=0.0001; ****, p<0.0001; n=4–5 groups, each of 10 flies). (E) Fraction of indicated flies showing PER to different concentrations of sucrose (ND represent standard fly food) (two-way ANOVA; ****, p<0.0001; n=4–5 groups, each of 10 flies). (F) hugin expression in the brain, illustrated by mCD8::GFP expression driven by huginGAL4. Scale bar represents 100 μm. (G) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons during the perfusion of glucose with or without TTX (one-way ANOVA; **, p=0.002; n=6–7). Horizontal black bar represents the duration of the indicated glucose solution stimulation. (H) AstA expression in the brain, illustrated by mCD8::GFP expression driven by AstAGAL4. Scale bar represents 100 μm. (I) Representative traces and quantification of ex vivo calcium responses of AstA+ neurons during the perfusion of glucose with or without TTX (one-way ANOVA; *, p=0.0187 for 50 mM glucose vs 50 mM glucose+TTX and p=0.022 for 50 mM glucose vs 20 mM glucose; n=6). (J) Representative images of pre-photoconversion (pre-PC) and post-photoconversion (post-PC) calcium-modulated photoactivatable ratiometric integrator (CaMPARI) signal in hugin-expressing neurons (upper). The red:green ratio represents intracellular Ca2+ concentrations (lower). Scale bar represents 100 μm (one-way ANOVA; ****, p<0.0001; n=18–23). One-way and two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.
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Figure 1—source data 1
Source data contain numerical values and statistical results for Figure 1.
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Figure 1—figure supplement 1
Satiety suppressed sweet sensitivity in a dopamine-independent manner.
(A) Representative traces (left) and quantification (right) of peak calcium transients of Gr5a+ neurons in indicated flies (t-test; *, p=0.0373; n=6). Horizontal black bar represents the duration of 5% sucrose stimulation. (B–D) Fraction of flies of the indicated genotypes showing PER to different concentrations of sucrose (two-way ANOVA; *, p=0.0498; ****, p<0.0001; B–C, n=4 groups, each of 10 flies). The area under the curve (AUC, D) represents sweet sensitivity in C. *p<0.05; ****p<0.0001. Student’s t-test and two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.
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Figure 1—figure supplement 1—source data 1
Source data contain numerical values and statistical results for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/108551/elife-108551-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
The effect of satiety signals on taste modulation.
(A) Fraction of fed flies of the indicated genotypes and at environmental temperatures showing proboscis extension reflex (PER) to different concentrations of sucrose (two-way ANOVA; *, p=0.012; ***, p=0.0002 for curve +>TrpA1 vs hugin >TrpA1 and p=0.0006 for the same curve at 400 mM; ****, p<0.0001; n=4 groups, each of 10 flies). (B) Fraction of starved flies of the indicated genotypes and environmental temperatures showing PER to different concentrations of sucrose (two-way ANOVA; *, p=0.0154; **, p=0.0081 for the curve CCHa1>TrpA1,TK>TrpA1 at 100 mM and CCHa2>TrpA1 at 200 mM, p=0.0011 for curve AstA >TrpA1 and LK>TrpA1 at 100 mM; ***, p=0.0005 for the curve hugin>TrpA1 at 100 mM and p=0.0001 for the curve CCHa2>TrpA1 at 800 mM; ****, p<0.0001; n=4–5 groups, each of 10 flies). Two-way ANOVA followed by post hoc test with Bonferroni correction was used for multiple comparisons when applicable.
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Figure 1—figure supplement 2—source data 1
Source data contain numerical values and statistical results for Figure 1—figure supplement 2.
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Figure 1—figure supplement 3
Activation of IPCs decreased the circulating sugar levels and increased sweet sensitivity.
(A) Hemolymph glucose levels from the indicated genotypes and treatment temperatures (t-test; ****, p<0.0001; n=8–9, 1 hr at 30°C). (B–C) Fraction of fed flies of the indicated genotypes and environmental temperatures showing proboscis extension reflex (PER) to different concentrations of sucrose (two-way ANOVA; **, p=0.0048; ****, p<0.0001; n=4 groups, each of 10 flies). Student’s t-test and two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.
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Figure 1—figure supplement 3—source data 1
Source data contain numerical values and statistical results for Figure 1—figure supplement 3.
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Figure 1—figure supplement 4
Sugar intake suppressed sweet sensation.
(A) Fraction of indicated flies which refed with different sugar with energy showing proboscis extension reflex (PER) to different concentrations of sucrose (two-way ANOVA; ***, p=0.0003; ****, p<0.0001; n=4 groups, each of 10 flies). (B) Fraction of indicated flies which refed with different sugar without energy showing PER to different concentrations of sucrose (two-way ANOVA; ****, p<0.0001; n=4 groups, each of 10 flies). (C) Fraction of indicated flies showing PER to different concentrations of fructose (two-way ANOVA; *, p=0.0255 for 12.5 mM and 25 mM; ***, p=0.0002; ****, p<0.0001; n=4 groups, each of 10 flies). (D) Fraction of indicated flies showing PER to different concentrations of trehalose (two-way ANOVA; **, p=0.0081 for 50 mM and p=0.0013 for 200 mM; ***, p=0.0002; ****, p<0.0001; n=4 groups, each of 10 flies). Two-way ANOVA followed by post hoc test with Bonferroni correction was used for multiple comparisons when applicable.
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Figure 1—figure supplement 4—source data 1
Source data contain numerical values and statistical results for Figure 1—figure supplement 4.
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Figure 1—figure supplement 5
Starvation led to a decrease in the circulating sugar levels.
Hemolymph glucose levels from indicated flies (one-way ANOVA; ****, p<0.0001; n=6–8). One-way ANOVA followed by post hoc test with Bonferroni correction was used for multiple comparisons when applicable.
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Figure 1—figure supplement 5—source data 1
Source data contain numerical values and statistical results for Figure 1—figure supplement 5.
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Figure 1—figure supplement 6
The response of different neuropeptide-expressing neurons to glucose.
(A) LK expression in the brain, illustrated by mCD8::GFP expression driven by LKGAL4. Scale bar represents 100 μm. (B) Representative traces and quantification of ex vivo calcium responses of LK+ neurons during the perfusion of glucose (n=6). (C–D) The distribution (C) and calcium responses (D) of TK+ neurons (n=6). (E–F) The distribution (E) and calcium responses (F) of CCHa1+ neurons (n=6). (G–H) The distribution (G) and calcium responses (H) of CCHa2+ neurons (n=6).
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Figure 1—figure supplement 6—source data 1
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Figure 1—figure supplement 7
The calcium response of hugin neurons upon glucose stimuli.
(A) Representative image of hugin neurons in the subesophageal zone (SEZ). Scale bar represents 50 μm. (B) Calcium responses of different clusters of hugin neurons during the perfusion of glucose (t-test, *, p=0.0151; n=6). Student’s t-test was used for comparisons.
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Figure 1—figure supplement 7—source data 1
Source data contain numerical values and statistical results for Figure 1—figure supplement 7.
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Figure 1—figure supplement 8
The calcium response of AstA+ neurons upon glucose treatment.
(A) Representative image of different parts of AstA neurons.Scale bar represents 50 μm. (B) Calcium responses of different clusters of hugin neurons during the perfusion of glucose (n=6).
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Figure 1—figure supplement 8—source data 1
Source data contain numerical values and statistical results for Figure 1—figure supplement 8.
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Figure 2
Glucose activated hugin+ neurons via Gltu1 and KATP channel.
(A) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons during the perfusion of glucose with or without phlorizin (1 mM), (t-test; **, p=0.0052; n=6–7). Horizontal black bar represents the duration of indicated glucose solution stimulation. (B) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons in indicated flies during the perfusion of glucose (t-test; *, p=0.022; n=6). (C) Fraction of flies of the indicated genotypes showing proboscis extension reflex (PER) to different concentrations of sucrose (two-way ANOVA; ***, p=0.0009 for FED 200 mM and 0.0001 for ST 200 mM and 400 mM; ****, p<0.0001; n=4 groups, each of 10 flies). (D–E) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons during the perfusion of glibenclamide (100 μM, D), glucose with alloxan (10 μM, E) (t-test; **, p=0.0091; n=6). (F) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons in indicated flies during the perfusion of glucose (t-test; **, p=0.0011; n=6). (G) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons during the perfusion of pyruvate (50 mM), (t-test; ns, p>0.05; n=6). (H) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons during the perfusion of D-glucose and L-glucose (50 mM), (t-test; **, p=0.0045; n=6). Student’s t-test and two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.
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Figure 2—source data 1
Source data contain numerical values and statistical results for Figure 2.
- https://cdn.elifesciences.org/articles/108551/elife-108551-fig2-data1-v1.xlsx
Figure 3 with 2 supplements
hugin+ neurons suppressed sweet taste through PK2-R1.
(A–B) Fraction of flies of the indicated genotypes and environmental temperatures showing proboscis extension reflex (PER) to different concentrations of sucrose (two-way ANOVA; *, p=0.043; ***, p=0.0004 for curve FED hugin>TrpA1 vs +>TrpA1 at 30°C, p=0.0009 for FED 400 mM at 30°C and p=0.0001 for ST 200 mM at 30°C; ****, p<0.0001; n=4–5 groups, each of 10 flies). (C) Fraction of flies showing PER to different concentrations of sucrose (two-way ANOVA; *, p=0.0486 for FED 50 mM and p=0.0438 for ST 12.5 mM; ***, p=0.0002 for FED 100 mM and p=0.0007 for ST 25 mM; ****, p<0.0001; n=4 groups, each of 10 flies). Flies were injected with saline or synthetic hugin for 30 min before the assay. (D) Representative traces (upper) and quantification (lower) of peak calcium transients of Gr5a+ neurons in indicated flies upon 5% sucrose after injection of synthetic hugin (t-test; ***, p=0.0047; n=6). Horizontal black bar represents the duration of sucrose stimulation. (E–F) Fraction of flies of the indicated genotypes showing PER to different concentrations of sucrose (two-way ANOVA; **, p=0.0055 for figure E and p=0.003 and 0.0016 for curve elav>PK2R2i vs +>PK2R2i and elav >PK2R2i v elav > +; ***,p=0.0002 for figure E ST 200 mM and p=0.0005 for figure F ST 200 mM; ****, p<0.0001; n=4 groups, each of 10 flies). Two-way ANOVA followed by post hoc test with Bonferroni correction was used for multiple comparisons when applicable.
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Figure 3—source data 1
Source data contain numerical values and statistical results for Figure 3.
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Figure 3—figure supplement 1
Knockout of PK2-R1 enhanced sweet sensation.
(A–C) Fraction of flies of the indicated genotypes showing proboscis extension reflex (PER) to different concentrations of sucrose (two-way ANOVA; (A) *, p=0.0464; **, p=0.001 for the curve w1118 vs PK2-R1 KO and p=0.0015 for the 400 mM; (B) *, p=0.0153; **, p=0.0038; ****, p<0.0001; (C) **, p0.0035; ***, p=0.0002; n=4 groups, each of 10 flies). Two-way ANOVA followed by post hoc test with Bonferroni correction was used for multiple comparisons when applicable.
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Figure 3—figure supplement 1—source data 1
Source data contain numerical values and statistical results for Figure 3—figure supplement 1.
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Figure 3—figure supplement 2
The expressing pattern of PK2-R1 in the proboscis.
Representative image of PK2-R1 in the proboscis. Scale bar represents 50 μm.
Figure 4 with 1 supplement
hugin+ neurons activated AstA+ neurons through PK2-R1.
(A–B) Fraction of flies of the indicated genotypes and environmental temperatures showing proboscis extension reflex (PER) to different concentrations of sucrose (two-way ANOVA; *, p=0.0153 for curve ST AstA >TrpA1 vs +>TrpA1 at 20°C and p=0.0237 for (B) 50 mM at 30°C; ****, p<0.0001; n=4–5 groups, each of 10 flies). (C) Co-localization (dashed box) of PK2-R1+ neurons (green) and AstA+ neurons (red) in the subesophageal zone (SEZ) region. Scale bar represents 100 μm. (D) Representative traces (upper) and quantification (lower) of peak calcium transients of AstA+ neurons after the photoactivation of hugin+ neurons (t-test; *, p=0.0114; n=6) from in vivo calcium imaging. Horizontal black bar represents the duration of red-light stimulation. (E–F) Fraction of flies of the indicated genotypes showing PER to different concentrations of sucrose (two-way ANOVA; *, p=0.0486 for curve FED AstA>PK2-R2i vs AstA >+ and p=0.0024 for FED 800 mM and p=0.033 for ST 100 mM and 200 mM; **, p=0.0043 and 0.0011 for AstA>PK2 R2 vs AstA >+ and +>PK2-R2i; ***, p=0.0008 for 25 mM and 50 mM and p=0.0001 for 200 mM; ****, p<0.0001; n=4 groups, each of 10 flies). Student’s t-test and two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.
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Figure 4—source data 1
Source data contain numerical values and statistical results for Figure 4.
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Figure 4—figure supplement 1
hugin+ neurons in the brain activated AstA+ neurons.
(A–B) Fraction of flies of the indicated genotypes and environmental temperatures showing proboscis extension reflex (PER) to different concentrations of sucrose (two-way ANOVA; (A) **, p=0.0082; ***, p=0.0007; ****, p<0.0001; (B) ***, p=0.0001; ****, p<0.0001; n=4 groups, each of 10 flies). Prior to the behavioral assay, the neural connection between the brain and the ventral nerve cord (VNC) was surgically severed, while the flies remained physically intact and retained normal proboscis musculature and motor output. PER was measured using the same protocol as in intact flies. (C) Representative traces (upper) and quantification (lower) of peak calcium transients of AstA+ neurons after the photoactivation of hugin+ neurons (t-test; *, p=0.0337; n=6) from ex vivo calcium imaging. Horizontal black bar represents the duration of red-light stimulation. Two-way ANOVA followed by post hoc test with Bonferroni correction was used for multiple comparisons when applicable.
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Figure 4—figure supplement 1—source data 1
Source data contain numerical values and statistical result Figure 4—figure supplement 1.
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Figure 5 with 2 supplements
AstA+ neurons inhibited Gr5a+ neurons through AstA-R1.
(A) Fraction of flies showing proboscis extension reflex (PER) to different concentrations of sucrose (two-way ANOVA; *, p=0.0219 for 50 mM; **, p=0.0072 for 25 mM and 100 mM and p=0.002 for 50 mM; ***, p=0.0002 for curve ST control peptide for AstA; ****, p<0.0001; n=4 groups, each of 10 flies). Flies were injected with saline or synthetic AstA for 30 min before the assay. (B) Representative traces (left) and quantification (right) of peak calcium transients of Gr5a+ neurons in indicated flies upon 5% sucrose after injection of synthetic AstA (t-test; *, p=0.0476; n=6). Horizontal black bar represents the duration of sucrose stimulation. (C) Localization of Gr5a+ neurons (green) and AstA+ neurons (red) in the brain. Scale bar represents 100 μm. (D) Colocalization of Gr5a+ neurons (green) and AstA-R1+ neurons (red). Arrows point to the neurons co-expressing the two receptors. Scale bar represents 50 μm. (E–F) Fraction of flies of the indicated genotypes showing PER to different concentrations of sucrose (two-way ANOVA; **, p=0.0088; ***, p=0.0004; ****, p<0.0001; n=4 groups, each of 10 flies). (G–H) Representative traces (left) and quantification (right) of peak calcium transients of Gr5a+ neurons in indicated flies upon 5% sucrose during the process of photoactivation of AstA+ neurons (G, n=6; t-test; **, p=0.0058) and hugin+ neurons (H, n=7–8; t-test; *, p=0.0351). Shadows represent the duration of red-light stimulation. Horizontal black bar represents the duration of sucrose stimulation. Student’s t-test and two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.
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Figure 5—source data 1
Source data contain numerical values and statistical results for Figure 5.
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Figure 5—figure supplement 1
The expressing pattern of AstA-R2 in the proboscis.
Representative image of AstA-R2 in the proboscis. Scale bar represents 50 μm.
Figure 5—figure supplement 2
Manipulating gene AstA-R1 enhances proboscis extension reflex (PER).
(A–C) Fraction of flies of the indicated genotypes showing PER to different concentrations of sucrose (two-way ANOVA; (A) ****, p0.0001; (B) *, p=0.404; **, p=0.0022; ***, p=0.0005; ****, p<0.0001; (C) **, p=0.0012 for the curve w1118 vs AstA-R1 KO and p=0.0019 for the 100 mM; n=4 groups, each of 10 flies). Two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.
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Figure 5—figure supplement 2—source data 1
Source data contain numerical values and statistical results for Figure 5—figure supplement 2.
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Figure 6 with 1 supplement
hugin-AstA circuitry inhibited feeding behavior.
(A) The illustration of experimental design in this figure. Note that all flies were maintained at 20°C before the feeding assay to prevent neuronal silencing by Shibirets1 during these procedures. (B) Fraction of flies of the indicated genotypes and environmental temperatures showing proboscis extension reflex (PER) to different concentrations of sucrose (two-way ANOVA; ***, p=0.0003; ****, p<0.0001; n=4 groups, each of 10 flies). (C–D) Food consumption of flies of the indicated genotypes and environmental temperatures (one-way ANOVA; **, p=0.004; ****, p<0.0001; n=8 groups, each of 4 flies). Food consumption is presented as fold change relative to the corresponding genetic control.
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Figure 6—source data 1
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Figure 6—figure supplement 1
hugin-AstA-Gr5a circuitry inhibited feeding behavior.
(A) Food consumption of flies of the indicated genotypes (one-way ANOVA; ***, p=0.0009; ****, p<0.0001; n=8 groups, each of 4 flies). (B) The TG content of indicated genotypes (one-way ANOVA; ***, p=0.0002; ****, p<0.0001; n=8 groups, each of 2 flies). (C) Food consumption of flies of the indicated genotypes (one-way ANOVA; ****, p<0.0001; n=8 groups, each of 4 flies). One-way ANOVA followed by post hoc test with Bonferroni correction was used for multiple comparisons when applicable.
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Figure 6—figure supplement 1—source data 1
Source data contain numerical values and statistical results for Figure 6—figure supplement 1.
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Figure 7 with 3 supplements
NMU+ neurons were the central energy sensor in mouse.
(A) Blood Neuromedin U (NMU) levels of starved mice refed with 20% sucrose (t-test; *, p=0.0373; n=6). (B) Two-bottle preference tests for starved mice refed with sucrose (t-test; ***, p=0.0002; n=7). (C) Two-bottle preference tests for starved mice with or without intraperitoneal injection of NMU peptide (YFLFRPRN-NH2, 4.5 μm/kg) (t-test; *, p=0.0293; n=7). (D) Two-bottle preference tests for indicated starved mice (t-test; **, p=0.0073; n=6). (E) Fiber photometry to record the calcium dynamics of NMU+ neurons in the ventromedial hypothalamus (VMH) with GCaMP6m (left) and representative IHC image of expressing GCaMP6m in VMH (right). (F–G) Representative trace (left) and heatmaps (right, n=5) showing calcium dynamics of NMU+ neurons in the VMH in response to gastric glucose infusion (20% sucrose, 200 μL), which elevates circulating glucose levels independently of oral sensory stimulation. (H) Experimental approach to assess calcium signaling in NMU+ neurons in the VMH in vitro (left) and representative IHC image of expressing GCaMP6m in VMH (right). (I–J) Representative traces and quantification of ex vivo calcium responses of NMU+ neurons during the perfusion of glucose with or without TTX (I), alloxan (J), and phlorizin (J) (one-way ANOVA; **, p=0.0049; ***, p=0.0001 for glucose+phlorizin and p=0.0006 for glucose+alloxan; n=6–7). Horizontal black bar represents the duration of indicated glucose solution stimulation. (K) Anterograde trans-synaptic tracing of downstream targets of NMU+ neurons. NMU+ neurons were labeled by GFP expression following injection of a Cre-dependent AAV2/1-DIO-GFP into NMU-Cre mice. This virus undergoes anterograde trans-synaptic transfer to postsynaptic neurons. To enable GFP expression specifically in downstream target regions, an AAV-Cre virus was locally injected into the rNST. As a result, postsynaptic neurons in the rNST receiving input from NMU+ neurons were labeled by GFP delivered anterogradely from upstream NMU+ neurons. Representative images show that GFP-labeled downstream neurons in the rNST colocalize with Calb2 immunoreactivity, indicating that NMU+ neurons preferentially target Calb2+ rNST neurons. (L) Fiber photometry to record the calcium dynamics of Calb2+ neurons in the rNST with GCaMP6m (left) and representative IHC image of expressing GCaMP6m in rNST (right). (M) Representative traces and quantification of calcium responses of Calb2+ neurons during glucose licking under physiological feeding conditions (500 mM sucrose), with or without NMU administration, assessing downstream modulation of sweet-responsive brainstem circuits (t-test; ****, p<0.0001; n=6). Student’s t-test and one-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.
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Figure 7—source data 1
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Figure 7—figure supplement 1
Neuromedin U (NMU) peptide suppressed sweet taste in fly.
(A–B) Fraction of flies showing proboscis extension reflex (PER) to different concentrations of sucrose (two-way ANOVA; (A) **, p=0.106; **, p=0.0025 for the curve H2O vs NMU and p=0.0015 for the 400 mM; (B) *, p=0.0106; **, p=0.0029; ****, p<0.0001; n=4 groups, each of 10 flies). Flies were injected with saline or synthetic NMU for 30 min before the assay. Two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.
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Figure 7—figure supplement 1—source data 1
Source data contain numerical values and statistical results for Figure 7—figure supplement 1.
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Figure 7—figure supplement 2
The expressing pattern of Neuromedin U (NMU) neurons and Calb2 neurons.
(A) Co-labeling of NMU+ neurons using a Cre-dependent mCherry reporter (red) and in situ hybridization (green). Scale bar represents 200 μm. (B) NMU arbors were extended near Calb2rNST neurons. Scale bar represents 200 μm. (C) Co-labeling of Calb2+ neurons with a Cre-dependent mCherry reporter (red) and NMU antibody staining (green). Scale bar represents 200 μm.
Figure 7—figure supplement 3
A working model.
Elevated circulating glucose following feeding directly activates hugin+neurons via cell-autonomous glucose sensing. Activated hugin neurons release hugin neuropeptide, which engages PK2-R1 receptors on AstA neurons, promoting AstA release. AstA then suppresses the sensitivity of Gr5a-expressing gustatory sensory neurons through AstA-R1 signaling, thereby reducing sweet-driven feeding behavior. Under starvation, reduced circulating glucose decreases endogenous hugin neuronal activity, partially relieving this inhibitory tone and permitting enhanced sweet sensitivity. However, experimental manipulation of the circuit demonstrates that the hugin-AstA axis retains the capacity to modulate feeding behavior across feeding states, indicating that it functions as a glucose-responsive, state-modulated inhibitory system rather than a strictly satiety-exclusive brake. The modest phenotype observed upon PK2-R2 reduction suggests the potential existence of parallel or complementary hugin-dependent pathways, possibly arising from functional heterogeneity within hugin neuronal subpopulations. These additional branches remain to be defined.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Drosophila melanogaster) | TH-KO | Other | Gift from Yi Rao | |
| Genetic reagent (D. melanogaster) | PK2-R1-KO | Other | Gift from Yi Rao | |
| Genetic reagent (D. melanogaster) | AstA-R1-KO | Other | Gift from Yi Rao | |
| Genetic reagent (D. melanogaster) | UAS-PK2-R1-RNAi | Bloomington Drosophila Stock Center | Cat: #29624 | |
| Genetic reagent (D. melanogaster) | UAS-PK2-R2-RNAi | Bloomington Drosophila Stock Center | Cat: #28781 | |
| Genetic reagent (D. melanogaster) | UAS-AstA-R1-RNAi | Bloomington Drosophila Stock Center | Cat: #27280 | |
| Genetic reagent (D. melanogaster) | UAS-Glut1-RNAi | TsingHua Fly Center | Cat: #3043 | |
| Genetic reagent (D. melanogaster) | UAS-Hex-C-RNAi | TsingHua Fly Center | Cat: #3684.N | |
| Genetic reagent (D. melanogaster) | AstA-Gal4 | Other | Gift from Yi Rao | |
| Genetic reagent (D. melanogaster) | LK-Gal4 | Other | Gift from Yi Rao | |
| Genetic reagent (D. melanogaster) | Hugin-Gal4 | Other | Gift from Yi Rao | |
| Genetic reagent (D. melanogaster) | TK-Gal4 | Other | Gift from Yi Rao | |
| Genetic reagent (D. melanogaster) | CCHa1-Gal4 | Other | Gift from Yi Rao | |
| Genetic reagent (D. melanogaster) | CCHa2-Gal4 | Other | Gift from Yi Rao | |
| Genetic reagent (D. melanogaster) | PK2-R1-Gal4 | Other | Gift from Yi Rao | |
| Genetic reagent (D. melanogaster) | TH-Gal4 | Other | Gift from Yi Rao | |
| Genetic reagent (D. melanogaster) | Elav-Gal4 | Bloomington Drosophila Stock Center | Cat: #25750 | |
| Genetic reagent (D. melanogaster) | Dilp2-Gal4 | Bloomington Drosophila Stock Center | Cat: #37516 | |
| Genetic reagent (D. melanogaster) | Gr5a-Gal4 | Bloomington Drosophila Stock Center | Cat: #57591 | |
| Genetic reagent (D. melanogaster) | Gr5a-LexA | Bloomington Drosophila Stock Center | Cat: #93014 | |
| Genetic reagent (D. melanogaster) | AstA-LexA | Other | Gift from Yi Rao | |
| Genetic reagent (D. melanogaster) | AstA-R1-LexA | Other | Gift from Yi Rao | |
| Genetic reagent (D. melanogaster) | UAS-TRPA1 | Other | Gift from David Anderson | |
| Genetic reagent (D. melanogaster) | UAS-shibirets | Other | Gift from David Anderson | |
| Genetic reagent (D. melanogaster) | UAS-GCaMP6m | Bloomington Drosophila Stock Center | Cat: #42748 | |
| Genetic reagent (D. melanogaster) | UAS-CD8-GFP, LexAop-CD2-RFP | Bloomington Drosophila Stock Center | Cat: #67093 | |
| Genetic reagent (D. melanogaster) | UAS-CaMPARI | Bloomington Drosophila Stock Center | Cat: #58761 | |
| Genetic reagent (D. melanogaster) | LexAop-Chrimson | Other | Gift from Wei Zhang | |
| Genetic reagent (D. melanogaster) | UAS-GCaMP6m; LexAop-CsChrimson | Other | Gift from Yufeng Pan | |
| Genetic reagent (Mus musculus) | NMU-KO | Cyagen | Cat: #S-KO-10720 | |
| Genetic reagent (M. musculus) | NMU-Cre | Shanghai Model Organisms Center | Cat: #NM-KI-200298 | |
| Genetic reagent (M. musculus) | Calb2-Cre | Shanghai Model Organisms Center | Cat: #NM-KI-200102 | |
| Antibody | Anti-Bruchpilot (Mouse monoclonal) | DSHB | Cat: # nc82 RRID:AB_2314866 | IF(1:100) (5 µL) |
| Antibody | Anti-GFP (Rabbit polyclonal) | Abcam | Cat: # G6556 RRID:AB_305564 | IF(1:500) (1 µL) |
| Antibody | Anti-dsRed (Rabbit polyclonal) | Clontech | Cat: #632496 RRID:AB_10013483 | IF(1:500) (1 µL) |
| Antibody | Anti-Calb2 (Rabbit polyclonal) | ImmunoStar | Cat: # 24445 RRID:AB_572223 | IF(1:500) (5 µL) |
| Antibody | Anti-mouse Alexa Fluor 488 | Thermo Fisher Scientific | Cat: #A11001 RRID:AB_2534069 | IF(1:500) (1 µL) |
| Antibody | Anti-rabbit Alexa Fluor 546 | Thermo Fisher Scientific | Cat: #A11010 RRID:AB_2534077 | IF(1:500) (1 µL) |
| Antibody | Anti-rabbit Alexa Fluor 488 | Thermo Fisher Scientific | Cat: #A11008 RRID:AB_143165 | IF(1:500) (1 µL) |
| Antibody | Anti-mouse Alexa Fluor 647 | Thermo Fisher Scientific | Cat: #A21235 RRID:AB_2535804 | IF(1:500) (1 µL) |
| Other | AAV2/9-EF1α-DIO-GCaMP6m-WPRE | Obio Biotechnology | Cat: #H4955 | Bacterial and virus strains |
| Other | AAV2/1-hsyn-EGFP-2A-Cre-WPRE | Obio Biotechnology | Cat: #H4942 | Bacterial and virus strains |
| Other | AAV2/1-hsyn-SV40 NLS-Cre | Brain Case | Cat: #BC-0159 | Bacterial and virus strains |
| Other | AAV2/9-EF1α-DIO-mcherry-WPRE | Brain Case | Cat: #BC-0016 | Bacterial and virus strains |
| Other | AAV2/9-EF1α-DIO-EGFP-WPRE | Brain Case | Cat: #BC-0015 | Bacterial and virus strains |
| Chemical compound | Brilliant Blue | MACKLIN | Cat: #E808678 | |
| Chemical compound | Phlorizin | TargetMol | Cat: #T2922 | |
| Chemical compound | Alloxan | TargetMol | Cat: #T7814L | |
| Chemical compound | Glibenclamide | TargetMol | Cat: #T1634 | |
| Chemical compound | All-trans-retinal | Sigma-Aldrich | Cat: #R2500 | |
| Chemical compound | L-Glucose | Sigma-Aldrich | Cat: #G5500 | |
| Chemical compound | Pyruvate | Sigma-Aldrich | Cat: #P5280 | |
| Chemical compound | Triton X-100 | Sigma-Aldrich | Cat: #T8787 | |
| Chemical compound | Calf serum | Thermo Fisher Scientific | Cat: #16010159 | |
| Commercial assays or kit | Glucose Content Assay Kit | Solarbio | Cat: #BC2500 | |
| Commercial assays or kit | NMU ELISA Assay Kit | BMASSY | Cat: #74030 | |
| Commercial assays or kit | NMU RNAscope | Pinpoease | Cat: #PIF2000 | |
| Software, algorithm | Fiji/ImageJ | NIH | ||
| Software, algorithm | GraphPad Prism 9 | GraphPad Software |
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Hugin-AstA circuitry is a novel central energy sensor that directly regulates sweet sensation in Drosophila and mouse
eLife 14:RP108551.
https://doi.org/10.7554/eLife.108551.4
