Thirst interneurons that promote water seeking and limit feeding behavior in Drosophila
- Quantitative and Systems Biology Graduate Program, UC, United States
- Department of Molecular and Cell Biology, UC, United States
Figures
Figure 1 with 1 supplement
Thirst behavior in an open field; a forward screen uncovers Durstig thirst neurons.
(A) Thirst-induced sequence of behavior. (B) Two-chambered open field assay with a vertical divider at the center. Thirsty flies avidly seek and occupy a discrete water source (dyed blue for …
Figure 1—figure supplement 1
Effects of activating neurons implicated in feeding, reward, and water learning on water seeking.
(A) Dopaminergic neuron activation effect on water seeking. (B) R48B04-Gal4, previously implicated in thirsty humidity preference, contains water seeking neurons that are different from those in …
Figure 2 with 4 supplements
Isolation of Janu thirst neurons from the Durstig pattern.
(A) Subtraction of R65D05 neurons (R65D05>Gal80) from the Durstig pattern decreases Durstig activation water seeking. One-way ANOVA/Dunnett's compared to Durstig>TrpA1. (B) Activation of R65D05 …
Figure 2—figure supplement 1
Characterization of R65D05-Gal4 neurons for thirst and hunger behaviors.
(A) Otd-nlsFLPo expresses Flippase predominantly in the central brain, effectively limiting R65D05-Gal4-driven GFP to the central brain. High gain to highlight absence of GFP positive neuron cell …
Figure 2—figure supplement 2
Characterization of R52A01-Gal4 neuron for thirst behaviors.
(A) Subtraction of subsets of neurons from Durstig>TrpA1 with patterns of GAL80 expression, occupancy of an inaccessible water source. Cha-Gal80: choline acetyltransferase enhancer, VGlut-Gal80: …
Figure 2—figure supplement 3
Janu neuron water seeking and feeding behavior characterization.
(A) Janu activation limited to central brain neurons promotes occupancy of open water source. One-way ANOVA/Dunnett's compared to Janu>UAS-STOP-TrpA1,otd-nlsFLPo. (B) Janu neuron inactivation …
Figure 2—figure supplement 4
Anatomy of individual Janu neurons.
Stochastic labeling of Janu neurons using a flip-out technique, detected with anti-FLAG (green), and counterstained with anti-VGAT (magenta). (A) Janu-AstA, arrowhead points to cell body (micrograph …
Figure 3 with 1 supplement
Janu-GABA SEZ local interneurons specifically promote water-seeking behaviors.
(A–D) Janu-GABA1 interneuron morphology in the SEZ of a Janu>multicolor flip-out brain, compressed z-stack. Arrowhead in (A) points to the cell body. Boxed area is enlarged in (B–D), showing …
Figure 3—figure supplement 1
Additional characterization of Janu-GABA neuron function in thirst behaviors.
(A) Decreased thirsty water seeking when VGAT is knocked down by a second, independent RNAi in Janu neurons. (B) No change in pharyngeal pumping rate with Gad1 knockdown in Janu neurons in thirsty …
Figure 4 with 1 supplement
The Janu-AstA neuron reciprocally regulates thirst and feeding behaviors.
(A–G) Janu-AstA neuron morphology and AstA expression. (A) Janu-AstA neuron morphology obtained from a multicolor flip-out brain. Green: anti-FLAG; magenta: anti-VGAT. Arrowhead points to cell body. …
Figure 4—figure supplement 1
Additional characterization of Janu-AstA neurons.
(A, B) Colocalization of Janu>myrGFP with AstA immunoreactivity, in the central brain (A) and in the ventral nervous system (B). Colocalization is limited to the Janu-AstA neurons. (C) AstA …
Figure 5 with 1 supplement
Thirst regulation of Janu neuron activity, and positive valuation of optogenetic activation of Janu.
(A) Examples of TRIC detection of neuronal activity (green) and the pattern of cells expressing the calcium indicator (magenta) under water replete and thirsty conditions in Janu neurons, detected …
Figure 5—figure supplement 1
Flies prefer activation of water seeking neurons.
(A) Positional preference of Janu > Chrimson flies without ATR. Positive values indicate attraction to optogenetic stimulation. (B) Positional preference for R65D05>Chrimson with and without ATR. (C)…
Figure 6 with 1 supplement
NPF neurons are postsynaptic to Janu-AstA neurons via the AstA-R2 receptor, and NPF inhibits water seeking.
(A) Janu-AstA presynaptic endings in boxed area imaged for immunohistochemistry. (B) Janu-AstA presynaptic endings partially overlap with NPF immunolabeling. Single 1 μm confocal section. Yellow …
Figure 6—figure supplement 1
Additional characterization of the role of NPF neurons in thirst behaviors.
(A) AstA-R1 receptor RNAi in NPF neurons had no effect on thirst or feeding behavior. (B) No change in pharyngeal pumping rate in thirsty NPF>AstA-R2 RNAi flies. (C) Immunohistochemical detection of …
Figure 7
Thirst interneuron circuitry in Drosophila.
(A) Functional relationship for thirst and feeding behavior. The dotted lines represent putative connectivity between interoceptive sensory neurons (ISN) and targets neuropeptide F (NPF) and Janu …
Tables
Author response table 1
| Figure | Question | Approach | Result | Interpretation |
|---|---|---|---|---|
| 3K | Are Janu-GABA neurons important for neural activation driven seeking? | We co-expressed a GABA neurotransmitter RNAi with TrpA1 activation. | Water seeking is suppressed. | Janu-GABA neuron activation drives replete water seeking. |
| 3L,M 3S1B,C | Are Janu-GABA neurons important for water ingestion? | We used well-established behavioral assays to specifically address ingestive behaviors. | Water ingestion does not change. But, the first bout of water intake decreases. Pumping rate is unchaged. | The first bout of water intake is mediated by Janu-GABA neurons. Other downstream neurons may be responsible for the maintenance of persistent ingestive behaviors. |
| 3N | Are Janu-GABA neurons important for fed and hungry feeding behavior? | We expressed a GABA neurotransmitter RNAi and assessed dry sucrose occupancy in fed and hungry flies. | Fed and hungry dry sucrose occupancy is unaltered. | Janu-GABA neurons are specific for thirst and do not contribute to feeding behavior. |
| 4L | Are Janu-AstA neurons important for neural activation driven seeking? | We co-expressed an AstA neurotransmitter RNAi with TrpA1 activation. | Water seeking is suppressed | Janu-AstA neuron activation drives replete water seeking. |
| 4M,N | Are Janu-AstA neurons important for water ingestion? | We used well-established behavioral assays to specifically address ingestive behaviors. | Water ingestion and first bout of water intake does not change. Pumping rate is unchanged. | Janu-AstA neurons are dispensable for thirsty water ingestion. |
| 5A,B,C | Are Janu-GABA and Janu-AstA activated by states of thirst and hunger? | We used TRIC, a calciumdependent fluorescent reporter, to assess neuronal activity. | Only the Janu-GABA1 subtype was highly active in thirsty flies. Janu-AstA was not active in thirsty or hungry states. | The Janu-GABA1 neuron is thirst activated. It may be regulated by the osmosensory ISNs. |
| 5D,E 5S1A,B,C | Does the activity of Janu and R65D05 neurons encode positive or negative valence? | We optogenetically activated Janu and R65D05 neurons to assess positional preference. | Flies exhibit positional preference for activation of Janu or R65D05 neurons. | Janu and R65D05 neurons encode positive valence. This is distinct from mammalian hunger and thirst neurons which encode negative valence. |
| 6EF 6S1B | Is AstA-R2 signaling on NPF neurons important for thirsty water ingestion? | We expressed an AstA-R2 RNAi in NPF neurons. We used well-established behavioral assays to specifically address ingestive behaviors. | Total water consumption and first bout water consumption are decreased. Pumping rate is unaffected | AstA signaling onto NPF neurons is important for water intake. |
| 6H | Is NPF signaling important for thirsty water seeking? | We expressed a NPF RNAi in thirsty flies to assess water seeking. | Water seeking is increased. | NPF released by NPF neurons suppresses water seeking. Thus, NPF can reciprocally modulate hunger and thirst. |
| 6I 6S1C | Are NPF neurons activated by thirst? | We used TRIC, a calcium-dependent fluorescent reporter, to assess neuronal activity. | Dorsomedial P1 and lateral L1 NPF neurons both are significantly activated in thirsty states. | NPF neurons encode thirst and are a critical convergence node for the integration of thirst and hunger signals. |
Additional files
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Supplementary file 1
InSITE strains and seeking index.
- https://cdn.elifesciences.org/articles/66286/elife-66286-supp1-v1.docx
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Supplementary file 2
Strains and resources.
- https://cdn.elifesciences.org/articles/66286/elife-66286-supp2-v1.xlsx
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Supplementary file 3
Experimental conditions and statistical analyses.
- https://cdn.elifesciences.org/articles/66286/elife-66286-supp3-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66286/elife-66286-transrepform-v1.docx
