Abstract
Hunger and thirst are ancient homeostatic drives for food and water consumption. Although molecular and neural mechanisms underlying these drives are currently being uncovered, less is known about how hunger and thirst interact. Here, we use molecular genetic, behavioral, and anatomical studies in Drosophila to identify four neurons that modulate food and water consumption. Activation of these neurons promotes sugar consumption and restricts water consumption, whereas inactivation promotes water consumption and restricts sugar consumption. By calcium imaging studies, we show that these neurons are directly regulated by a hormone signal of nutrient levels and by osmolality. Finally, we identify a hormone receptor and an osmolality-sensitive ion channel that underlie this regulation. Thus, a small population of neurons senses internal signals of nutrient and water availability to balance sugar and water consumption. Our results suggest an elegant mechanism by which interoceptive neurons oppositely regulate homeostatic drives to eat and drink.
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🧬 Organisms
💻 Software
✨ Fluorophores
🧪 Sample Preparation
📷 Detectors
💻 Software Details
🏛️ Research Organizations (ROR)
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📋 Methods
Additional details are available in Extended Experimental Procedures. Blue Dye Consumption experiments Flies (15-20/vial) were transferred from food to filter paper soaked in 200mM sucrose and blue dye. After feeding animals were scored a 0 (no dye in abdomen), 1 (less than half of the abdomen was blue), or 2 (half or more of the abdomen contained dye). The scores of all flies in one vial were averaged and considered one trial.
Proboscis Extension Response Assays
PER was performed as described ( Marella et al., 2012 ), except that each animal was considered a data point, and was categorized as responding 0, 1, 2, or 3 times.
Temporal Consumption Assays
Assays were performed as described ( Pool et al., 2014 ). Animals were presented with a taste stimulus ten times and total consumption time was monitored. To generate thirsty flies, flies were placed in a sealed chamber with ~250g CaSO 4 (Drierite, stock# 23001) for 2 hours, unless otherwise noted.
Immunohistochemistry
Immunohistochemistry was performed as described ( Marella et al., 2012 ).
Calcium and voltage imaging
GCaMP5G or GCaMP6s imaging of taste responses was performed as described ( Harris et al., 2015 ). Stacks of 14-20 Z-slices were collected at approximately 0.3 Hz. For GCaMP5G or GCaMP6s imaging of AKH or osmolality responses, the brain was removed in ice-cold calcium and magnesium-free artificial hemolymph-like solution (AHL), transferred to a perfusion chamber with room temperature AHL, and immobilized with tungsten wire. Image analysis was performed in ImageJ.
Hemolymph osmolality measurements
An osmometer was assembled and used as described ( Arav and Rubinsky, 1994 ), with minor modifications detailed in Extended Experimental Procedures.
Show full methods section
Additional details are available in Extended Experimental Procedures. Blue Dye Consumption experiments Flies (15-20/vial) were transferred from food to filter paper soaked in 200mM sucrose and blue dye. After feeding animals were scored a 0 (no dye in abdomen), 1 (less than half of the abdomen was blue), or 2 (half or more of the abdomen contained dye). The scores of all flies in one vial were averaged and considered one trial.
Proboscis Extension Response Assays
PER was performed as described ( Marella et al., 2012 ), except that each animal was considered a data point, and was categorized as responding 0, 1, 2, or 3 times.
Temporal Consumption Assays
Assays were performed as described ( Pool et al., 2014 ). Animals were presented with a taste stimulus ten times and total consumption time was monitored. To generate thirsty flies, flies were placed in a sealed chamber with ~250g CaSO 4 (Drierite, stock# 23001) for 2 hours, unless otherwise noted.
Immunohistochemistry
Immunohistochemistry was performed as described ( Marella et al., 2012 ).
Calcium and voltage imaging
GCaMP5G or GCaMP6s imaging of taste responses was performed as described ( Harris et al., 2015 ). Stacks of 14-20 Z-slices were collected at approximately 0.3 Hz. For GCaMP5G or GCaMP6s imaging of AKH or osmolality responses, the brain was removed in ice-cold calcium and magnesium-free artificial hemolymph-like solution (AHL), transferred to a perfusion chamber with room temperature AHL, and immobilized with tungsten wire. Image analysis was performed in ImageJ.
Hemolymph osmolality measurements
An osmometer was assembled and used as described ( Arav and Rubinsky, 1994 ), with minor modifications detailed in Extended Experimental Procedures.
Electrophysiology
Extracellular recordings were performed in live animals as described ( Pool et al., 2014 ).
Statistical Analyses
Student’s t-test, with Sidak correction for multiple comparisons, was used to compare two groups. ANOVA followed by Tukey’s post hoc test was used to compare three or more groups. ANOVA followed by Dunnet’s post hoc test was used to compare multiple responses of varying stimuli.
Supplementary Material 1 2 3 4 5 tab1
📊 Figures
Figure 1
Identification of neurons that promote sucrose consumption
A. Behavioral screen for flies that overconsume sucrose. Gal4 lines were crossed to UAS-dTRPA1 for heat-inducible neural activation and tested for sucrose consumption at 30u00b0C under non-deprived co...
Figure 2
ISNs respond to AKH and are inhibited by insulin
A. (left) Expression of AKHR-Gal4, UAS-mCD8::GFP in brain. (second) AKHR-Gal4, UAS-mCD8::RFP in SEZ, (third) R34G02-LexA, lexAop-mCD8::GFP in SEZ, (right) overlay. Scale 50 u03bcm. B. (left) GCaMP5G i...
Figure 3
Identification of nanchung and its role in water consumption
A. Behavioral screen for genes regulating water consumption. UAS-RNAi lines were crossed to nSyb-Gal4 . 10 RNAi and 10 sibling control flies were assayed/RNAi line. Average water consumption time in R...
Figure 4
Nanchung SEZ neurons respond to osmolality
A. nan-Gal4, UAS-GCaMP6s expression in brain (left) and example u0394F/F heat-map of the same brain (right) upon an extracellular osmolality decrease of 200mOsm/kg. Scale 50 u03bcm. B. u0394F/F traces...
Figure 5
Nanchung SEZ neurons are ISNs
A. Co-expression of R34G02-LexA and nan-Gal4 in ISNs. Zoom shows one nan-Gal4 cluster. Scale 20 u03bcm. B. (left) Low osmolality response and (right) AKH response in the same cell. C. maximum u0394F/F...
Figure 6
Drosophila hemolymph osmolality decreases during starvation
A. Top: Temperature (Temp.) gradient osmometer. Distance between sample and standard ice/liquid interfaces was used to calculate sample osmolality. Bottom: Freezing interfaces (white dots) from standa...
Figure 7
ISNs promote sucrose consumption and inhibit water consumption
A. ISN activation with dTRPA1 alters water and 1M sucrose consumption. Desiccated (des.), hydrated (hyd.). n=30-76 flies. Water consumption: ***p<0.001, t-tests (23u00b0C vs 32u00b0C) with Holm-Sid...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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